Split Inteins, Conjugates and Uses Thereof

ABSTRACT

Disclosed herein are split inteins, fused proteins of split inteins, and methods of using split inteins to efficiently purify and modify proteins of interest.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This invention was made with U.S. government support under Grant No.GM086868 awarded by the National Institutes of Health (NIH). The U.S.government has certain rights in the invention.

This application contains, as a separate part of disclosure, a SequenceListing in computer-readable form (filename: 47046A_SeqListing.txt;547,838 bytes—ASCII text file—created Jun. 20, 2013) which isinconcorporated by reference in its entirety.

BACKGROUND

Protein splicing is a post-translational process catalyzed by a familyof proteins known as inteins.(1) During this process, an intein domaincatalyzes its own excision from a larger precursor protein andsimultaneously ligates the two flanking polypeptide sequences (exteins)together. While most inteins catalyze splicing in cis, a small subset ofthese proteins exist as naturally fragmented domains that are separatelyexpressed but rapidly associate and catalyze splicing in trans. Giventheir capacity to make and break polypeptide bonds (inteins can beconsidered protein ligases), both cis and trans-splicing inteins havefound widespread use as chemical biological tools.(2)

Despite the growing use of inteins in chemical biology, their practicalutility has been constrained by two common characteristics of thefamily, namely (i) slow kinetics and (ii) context dependent efficiencywith respect to the immediate flanking extein sequences.(3,4) Recently,a split intein from the cyanobacterium Nostoc punctiforme (Npu) wasshown to catalyze protein trans-splicing on the order of a minute,rather than hours like most cis- or trans-splicing inteins.(5)Furthermore, this intein was slightly more tolerant of sequencevariation at the critical +2 C-extein residue than other inteins.(6)

Thus, a need exists for more robust and more efficient split inteins foruse in a variety of protein purification and protein modificationapplications.

SUMMARY

Disclosed are split intein N- and C-fragments, variants thereof, andmethods of using these split inteins in polypeptide purification andmodification.

Thus, provided herein are fusion proteins of a polypeptide and a splitintein N-fragment, or variant thereof, as described below in greaterdetail. Also provided are complexes of the fusion protein and a splitintein C-fragment or variant thereof as described in detail below. Thecomplex of the fusion protein and C-fragment or variant thereof can bevia a covalent interaction between the fusion protein and C-fragment orvariant or via a non-covalent interaction (e.g., ionic, H-bonding,and/or van der Waals interaction).

Further provided herein are split intein C-fragments or variantsthereof. In some cases the split intein C-fragment further comprises alinker, such as a peptide linker, or other linkers as described below indetail. A specific peptide linker contemplated is −SGGC (SEQ ID NO: 705)attached to any of the split intein C-fragments described below. Thelinker can be tailored so as to allow for attachment of a split inteinC-fragment of interest to a support, e.g., a bead, a resin, a slide, aparticle.

Also provided herein are methods using the split intein N- andC-fragments, or variants thereof, as described in detail below. Moreparticularly, provided herein are methods comprising (a) contacting (1)a fusion protein comprising a polypeptide and a split intein N-fragment,or a variant thereof, as described in detail below and (2) a splitintein C-fragment or a variant thereof, as described in detail below;wherein contacting is performed under conditions that permit binding ofthe split intein N-fragment to the split intein C-fragment to form anintein intermediate; and (b) contacting the intein intermediate with anucleophile to form a conjugate of the protein and the nucleophile. Invarious embodiments, the split intein C-fragment, or variant thereof, isbound to a support. In some embodiments, the support is a bead, a resin,a particle or a slide. It will be appreciated that selection of theN-fragment and C-fragment can be from the same wild type split intein(e.g., both from Npu, or a variant of either the N- or C-fragment asdiscussed in great detail below), or alternatively can be selected fromdifferent wild type split inteins or the consensus split inteinsequences discussed below, as it has been discovered that the affinityof a N-fragment for a different C-fragment (e.g., Npu N-fragment orvariant thereof with Ssp C-fragment or variant thereof) still maintainssufficient binding affinity for use in the disclosed methods. Moreover,such a finding allows for a single C-fragment or variant thereof boundto a support to be useful in purification and/or modification methodsdisclosed herein with a fusion protein wherein the N-fragment is any ofthe ones disclosed herein, or a variant thereof. Thus, one can select anN-fragment that has advantages for any individual polypeptide ofinterest, e.g., one that expresses better than others disclosed herein.

The fusion protein can be in a whole cell lysate or secreted from a cell(e.g., a mammalian cell) and in a cell supernatant. In some cases, thepolypeptide of the fusion protein is an antibody, e.g., an IgG antibody.In some embodiments, the N-fragment is fused to one or both of the heavychains of the antibody. In some embodiments, the N-fragment is fused toone or both of the light chains of the antibody. The methods disclosedherein can further comprise washing the intein intermediate (prior tocontact with the nucleophile) to remove the cell lysate or cellsupernatant, for example.

The methods disclosed herein can further comprise isolating theresulting conjugate of the polypeptide and nucleophile. Thus, themethods disclosed herein can be useful as an efficient purification forpolypeptides prepared by recombinant protein methods.

The nucleophile can be a thiol to form a conjugate that is anα-thioester of the polypeptide. In some cases, the resulting α-thioestercan be further modified by contacting with a second nucleophile,employing the well known α-thioester chemistry for protein modification.In some cases, the methods disclosed herein can provide conjugates ofthe polypeptide, which in some cases is an antibody (e.g., an IgGantibody), and a nucleophile (e.g., a drug, a polymer, anoligonucleotide).

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows trans-splicing of split DnaE inteins. (a) Scheme depictingprotein trans-splicing of the KanR protein with a variable localC-extein sequence. (b) In vivo relative trans-splicing efficiencies at30° C. with the endogenous “CFN” C-extein sequence and exogenous “CGN”,“CEN”, and “CRN” sequences. IC₅₀ values (±SE, n=3-4) are normalizedrelative to that of intact KanR proteins with the appropriate C-exteintri-peptide.

FIG. 2 shows in vitro half-lives of trans-splicing reactions. Indicatedsplit intein pairs fused to model exteins Ub or SUMO (Ub-Int^(N) andInt^(C)-SUMO) were mixed at either 30° C. or 37° C., and the formationof products was monitored over time by gel electrophoresis. (a)Half-lives were extracted from the reaction progress curves fit to astandard first-order rate equation (±SE, n=3). Representativecoomassie-stained SDS-PAGE gels showing (b) fast Ava splicing at 37° C.and (c) inefficient Ssp splicing at 37° C.

FIG. 3 shows sequence-activity relationships in split DnaE inteins. (a)Inteins in order of in vivo splicing activity with selected slices fromthe corresponding multiple sequence alignment. (b) Rendering of the Npustructure highlighting the proximity of position 120 to the terminalcatalytic residues C1 and N137. (c) In vivo analysis of the C120Gmutation in the Aha intein (±SD, n=3). (d) Rendering of the Npustructure highlighting key catalytic residues (sticks) and importantnon-catalytic positions (spheres) that modulate Ssp activity. (e) Invivo analysis of Ssp-to-Npu point mutations that improve Ssp activity(±SD, n=4). Note that all residue numberings correspond to the relevantpositions on Npu as defined by the NMR structure (PDB: 2KEQ).(21)

FIG. 4 shows engineered versions of ultrafast DnaE inteins supportefficient expressed protein ligation. (a) Scheme depicting the formationof the linear thioester intermediate and its use to generate a proteinα-thioester for EPL. (b) Coomassie-stained SDS-PAGE gel depictingefficient MESNa thiolysis of ubiquitin from a fused AvaDnaE intein toyield the Ub-MES thioester, 4. (c) Fluorescent SDS-PAGE gels showing theformation of the Ub-CGK(Fluorescein) ligated product (6) from one-potthiolysis and native chemical ligation reactions using the inteinsindicated. (d) Reverse phase HPLC chromatographs showing pH dependenceof the relative populations of precursor amide (1) and linear thioester(2).

FIG. 5 shows sequence alignments of split DnaE inteins. Numberingfollows that of Npu as assigned for the NMR structure (PDB 2KEQ).Critical catalytic residues are marked with an asterisk.

FIG. 6 shows sequence logos for high- and low-activity inteins. Inteinsare ranked based on in vivo activity with a “CFN” C-extein sequence. Thehigh and low activity inteins are distinguished based on a cut-off IC₅₀value of 350 μg/mL of kanamycin, and the Aha intein is included in thehigh-activity set, given that the C¹²⁰G mutation dramatically restoreshigh activity.

FIG. 7 shows purification of C-terminal α-thioesters usingsplit-inteins. A) Scheme of the split-intein based purification ofprotein C-terminal α-thioesters. B) Sequence of WT Npu^(C) and itsmutant Npu^(C)-AA having a linker to immobilize it onto a solid support(underlined). For the thiolysis experiments in solution the C-terminalCys residue was previously alkylated with iodoacetamide.

FIG. 8 shows Purification of soluble protein α-thioesters using theNpu^(C)-AA affinity column. A) Scheme of the purification strategy usingsplit Npu DnaE intein. B) Purification of Ub-thioester (Ub-COSR) fromcell lysates. C) Purification of MBP-thioester (MBP-COSR) from celllysates. Both purifications were monitored by SDSPAGE analysis stainedwith Coomassie (top) or Western Blot using an α-His antibody.

FIG. 9 shows RP-HPLC and MS analysis of Ub and MBP α-thioesters. A)RP-HPLC (top) and MS (bottom) analysis of Ub-COSR eluted from theNpu^(C)-AA column. B) RP-HPLC (top) and MS (bottom) analysis of MBP-COSReluted from the Npu^(C)-AA column.

FIG. 10 shows the effect of the −1 residue on the efficiency of theon-resin thiolysis. 20 different Ub-Npu^(N) proteins were expressedcontaining each of the 20 proteinogenic amino acids at the C-terminus ofUb (−1 residue) and purified over Npu^(C)-AA columns. Cleavage yieldsfrom the Npu^(C)-AA column were estimated by gel elctrophoresis andamounts of thioester versus side reactions (mainly hydrolysis) weredetermined by RP-HPLC and MS analysis.

FIG. 11 shows purification of H2B(1-116)-α-thioester under denaturingconditions. A) SDSPAGE analysis of the purification of H2B(1-116)α-thioester over the Npu^(C)-AA column in the presence of 3 M urea.RP-HPLC (B) and MS (C) analysis of E1 from panel A confirmed thepresence of the desired H2B thioester.

FIG. 12 shows purification of αDEC thioesters expressed in 293T cellsusing the split-intein column. A) Expression levels of αDEC fused todifferent inteins in 293T cells. B) Purification of αDEC α-thioesterthrough the Npu^(C)-AA affinity column. C) Expressed Protein Ligation(EPL) of αDEC-thioester with an N-terminal Cys containing fluorescentpeptide.

FIG. 13 shows EPL directly using Int^(C)-column eluted thioesters.RP-HPLC (30-73% B gradient, 214 nm and 440 nm detection) and MS analysisof the reactions between the H-CGK(Fl)-NH₂ peptide and MBP (A) and PHPT1(B) MES thioesters, purified from E. coli using the Int^(C)-column.

FIG. 14 shows a one-pot purification/ligation experiment of ubiquitin tothe H-CGK(Fluorescein)-NH₂ peptide (CGK(Fl)). Ub-Npu^(N) from E. colicell lysates was bound to the Int^(C)-column, and after removal ofcontaminants through extensive washes, intein cleavage and ligation weretriggered by addition of 200 mM MES and 1 mM CGK(Fl) peptide. Coomassiestained SDS-PAGE analysis and in gel fluorescence of thepurification/ligation (left). RP-HPLC (detection at 214 and 440 nm) andESI-TOF MS (right) of the eluted fractions confirms the desired ligatedprotein was obtained in one step directly from cell lysates with aligation yield close to 95% (quantified by RP-HPLC).

FIG. 15 shows the semi-synthesis of H2B-K120Ac under denaturingconditions. A) Coomassie stained SDS-PAGE analysis of H2B(1-116)α-thioester generation in the presence of 2 M urea (sup: cell lysatesupernatant, trit: 1% triton wash of the inclusion bodies, inp:solubilized inclusion bodies used as input for the Int^(C)-column).E1-E6 were pooled, concentrated to 150 μM and ligated to the peptideH-CVTK(Ac)YTSAK-OH at 1 mM for 3 h at r.t. B) RP-HPLC (left) of theligation reaction mixture and MS (right) of the ligated H2B-K120Acproduct.

FIG. 16 shows the characterization of αDEC205 ligated to theH-CGK(Fluorescein)-NH2 peptide (CGK(Fl)). Elution fractions from theNpu^(C)-column containing αDEC205-MES thioester were concentrated to 20μM and ligated to the CGK(Fl) fluorescent peptide at 1 mM for 48 h atr.t. A) ESI-TOF MS analysis of degycosylated and fully reduced HC afterligation, showing 75% of the HC are labeled. Expected mass for ligationproduct=50221.2 Da. Free HC=49575.0 Da. B) SEC-MALS analysis of theligated antibody showing that it retains its tetrameric structure afterthiolysis and ligation (MW=151 kDa, MW calc=148 kDa). C) Binding ofαDEC205-CGK(Fl) to the DEC205 receptor. Dose dependent binding ofαDEC205-CGK(Fl) (left) or a control α-DEC205 antibody (right) to CHOcells expressing the mouse DEC205 receptor monitored by flow cytometryusing a PE labeled α-mouse IgG. Binding to control CHO/NEO cells, whichdon't express the receptor is shown in gray.

FIG. 17 shows purification of αDEC thioesters expressed in CHO cellsusing a split-intein column. Top) Coomassie stained SDSPAGE gel of thepurification of αDEC-MES thioester from CHO cells using aNpu^(C)-column. Bottom) Western blot analysis of the same purification.

FIG. 18 shows purification of αDEC thioesters using an Ava^(C)split-intein column and Western blot analysis of the purification ofαDEC thioesters from mammalian cell supernatants using anAva^(C)-column.

FIG. 19 shows expression tests of αDEC205 antibody fused to Ava^(N)split intein through the C-terminus of the antibody light chain andwestern blot analysis of CHO cell supernatants expressing theαDEC205-AvaN fusion at different timepoints.

DETAILED DESCRIPTION

Of the roughly 600 inteins currently catalogued, (7) less than 5% aresplit inteins, mostly from a family known as the cyanobacterial splitDnaE inteins (8). Surprisingly, only six of these, including Npu, havebeen experimentally analyzed to any extent, (6,9,10) and only Npu andits widely-studied, low-efficiency ortholog from Synechocystis speciesPCC6803 (Ssp) have been rigorously characterized in vitro.(5,11)

A rapid survey of 18 split DnaE inteins was performed using an in vivoscreening method to accurately compare the efficiencies of splitinteins(12,13) In this assay, the two fragments of a split intein areco-expressed in E. coli as fusions to a fragmented aminoglycosidephosphotransferase (KanR) enzyme. Upon trans-splicing, the active enzymeis assembled, and the bacteria become resistant to the antibiotickanamycin (FIG. 1a ). More active inteins confer greater kanamycinresistance and thus have a higher IC₅₀ value for bacterial growth as afunction of kanamycin concentration. This assay can be carried out inthe background of varying local C-extein sequences without significantlyperturbing the dynamic range. Since all DnaE inteins splice the samelocal extein sequences in their endogenous context, this screen wasoriginally carried out in a wild-type C-extein background (CFN) withinthe KanR enzyme. As expected, bacteria expressing the Npu intein had ahigh relative IC₅₀, whereas clones expressing Ssp showed poor resistanceto kanamycin. Remarkably, more than half of the DnaE inteins showedsplicing efficiency comparable to Npu in vivo at 30° C. (FIG. 1b ).

To confirm that the high IC₅₀ values observed in vivo reflected rapidtrans-splicing, a series of kinetic studies were performed understandardized conditions in vitro. For this, individually expressed andpurified split DnaE intein fragments fused to model N- and C-exteindomains, ubiquitin and SUMO were made. The endogenous local exteinresidues were preserved as linkers between the extein domains and inteinfragments to recapitulate a wild-type-like splicing context. Cognateintein fragments were mixed at 1 μM, and the formation of the Ub-SUMOspliced product at 30° C. and 37° C. was monitored by gelelectrophoresis. These assays validated that the new inteins withhigh-activity in vivo could catalyze trans-splicing in vitro in tens ofseconds, substantially faster than Ssp (FIG. 2a ). Interestingly, all ofthe inteins analyzed except Ssp showed increased splicing rates at 37°C. Furthermore, all of the fast-splicing inteins showedlow-to-undetectable levels of side reactions (FIG. 2b ), again incontrast to Ssp (FIG. 2c ).

The tolerance of the split inteins to C-extein sequence variation wasinvestigated. Previously, the sensitivity of DnaE inteins was noted tochanges at the +2 position in the C-extein.(6,12) Thus, all the splitDnaE inteins were analyzed in the presence of a +2 glycine (CGN),glutamic acid (CEN), or arginine (CRN) in the in vivo screening assay(FIG. 1b ). Like Npu and Ssp, most of the inteins showed a dramaticdecrease in activity in the presence of all three +2 mutations. Of thetested amino acids, glutamic acid was tolerated best for every intein,suggesting a conserved mechanism for accommodating a negative charge atthis position. To more accurately assess the magnitude of the effect ofC-extein mutations on trans-splicing, the Npu, Cra(CS505), and Cwainteins were analyzed in vitro in the presence of a +2 glycine. Allthree of these reactions were characterized by rapid accumulation ofthioester intermediates, which slowly resolved over tens of minutes intothe spliced product and the N-extein cleavage product. Consistent withpreviously reported observations, these data indicate that split DnaEinteins require steric bulk at the +2 position for branched intermediateresolution and efficient splicing.(12) It is noteworthy that theCra(CS505) and Cwa inteins showed greater C-extein promiscuity in vivo,while Ssp(PCC7002) did not tolerate any of the mutations tested. Thisdemonstrates that subtle sequence variation between split inteins canafford differential promiscuity. Thus, this property may be furtheroptimized through directed evolution(12) or rational design.

These data indicate that the split DnaE inteins are highly divergent inactivity, despite all having evolved to catalyze trans-splicing onvirtually identical substrates. Interestingly, the key catalyticresidues involved in splicing are conserved across the entire family(FIG. 5). Thus, residues that affect splicing activity are non-catalyticand perhaps only moderately conserved. The measurements of relativeactivity can facilitate the discovery of specific sequence features thatdifferentiate high-activity inteins from inefficient ones. Indeed,sequence homology analysis indicates that inteins with high activity aremore homologous to one another than they are to the low-activityinteins. One significant outlier to this observation is the intein fromAphanothece halophytica (Aha), which despite having greater than 65%sequence identity to the high-activity inteins, was inactive with thewild-type “CFN” C-extein motif in vivo. Closer inspection of a multiplesequence alignment indicated that this intein has a non-catalyticcysteine (position 120) in place of an otherwise absolutely conservedglycine (FIG. 3a ). Furthermore, this position is close to the inteinactive site, where an extra nucleophile may facilitate undesirable sidereactions (FIG. 3b ). Gratifyingly, mutating this cysteine to glycinereinstated high activity in the Aha intein whilst the reverse mutationdestroyed the splicing activity of Npu (FIG. 3c ), validating thepredictive capacity of these data.

Further analysis of the split intein sequence alignment indicated thatseveral positions have strong amino acid conservation amongst thehigh-activity inteins but diverge for the low-activity inteins (FIGS. 3a, 6). These may be sites where the fast inteins have retained beneficialinteractions that have been lost in slow ones. To test this idea,several positions were chosen where this sequence-activity correlationwas apparent and replaced the residue in Ssp with the correspondingamino acid found in the fast inteins. Consistent with this hypothesis,several point mutations increased the activity of Ssp in vivo (FIGS. 3e). While the specific roles of these residues are not explicitly clear,especially given that they lie outside of the active site (FIG. 3d ),their locations on the intein fold (14) may provide some insights intotheir function. For example, at position 56, an aromatic residue ispreferred in the high-activity inteins. This position is adjacent to theconserved catalytic TXXH motif (positions 69-72), and an aromaticresidue may facilitate packing interactions to stabilize those residues.Similarly, a glutamate is preferred at position 122, proximal tocatalytic histidine 125. The glutamate at position 89 is involved in anintimate ion cluster that was previously shown to be important forstabilizing the split intein complex.(13) Interestingly, E23 is distantfrom the catalytic site and has no obvious structural role. Thisposition is conceivably important for fold stability or dynamics as haspreviously been observed for activating point mutations in otherinteins.(15,16)

The discovery of new, fast trans-splicing inteins has broad implicationsfor protein chemistry. Indeed, the discovery of Npu fueled a resurgencein the use of split intein-based technologies.(13,17,18) While no singleintein may be ideal for every protein chemistry endeavor, theavailability of several new fast-splicing split inteins can provideoptions to enhance the efficiency of most trans-splicing applications.For example, one common problem in working with split inteins is lowexpression yield or poor solubility of an intein fragment fusion to aprotein of interest. Indeed, the over-expression and purificationefforts here show that the Ub-IntN and IntC-SUMO fusions have markedlydifferent yields of soluble expression, depending on the intein. Thus, ashort-list of highly active split inteins with varying behavior willserve as starting point for empirical optimization of a giventrans-splicing application.

Furthermore, the fragments of the different fast-splicing split inteinscan be mixed as non-cognate pairs and still retain highly efficientsplicing activity, further expanding the options available for anytrans-splicing application. For example, the N-fragment split intein ofNpu or variant thereof can bind to the C-fragment of Npu or variantthereof or any of the other split intein C-fragments or variantsdiscussed below. Similarly, the N-fragment of Ssp or variant thereof canbind to the C-fragment of Ssp or variant thereof or any of the othersplit intein C-fragments or variants thereof discussed below; theN-fragment of Aha or variant thereof can bind to the C-fragment of Ahaor variant thereof or any of the other split intein C-fragments orvariants thereof discussed below; the N-fragment of Aov or variantthereof can bind to the C-fragment of Aov or variant thereof or any ofthe other split intein C-fragments or variants thereof discussed below;the N-fragment of Asp or variant thereof can bind to the C-fragment ofAsp or variant thereof or any of the other split intein C-fragments orvariants thereof discussed below; the N-fragment of Ava or variantthereof can bind to the C-fragment of Ava or variant thereof or any ofthe other split intein C-fragments or variants thereof discussed below;the N-fragment of Cra(CS5505) or variant thereof can bind to theC-fragment of Cra(CS5505) or variant thereof or any of the other splitintein C-fragments or variants thereof discussed below; the N-fragmentof Csp(CCY0110) or variant thereof can bind to the C-fragment ofCsp(CCY0110) or variant thereof or any of the other split inteinC-fragments or variants thereof discussed below; the N-fragment ofCsp(PCC8801) or variant thereof can bind to the C-fragment ofCsp(PCC8801) or variant thereof or any of the other split inteinC-fragments or variants thereof discussed below; the N-fragment of Cwaor variant thereof can bind to the C-fragment of Cwa or variant thereofor any of the other split intein C-fragments or variants thereofdiscussed below; the N-fragment of Maer(NIES843) or variant thereof canbind to the C-fragment of Maer(NIES843) or variant thereof or any of theother split intein C-fragments or variants thereof discussed below; theN-fragment of Mcht(PCC7420) or variant thereof can bind to theC-fragment of Mcht(PCC7420) or variant thereof or any of the other splitintein C-fragments or variants thereof discussed below; the N-fragmentof Oli or variant thereof can bind to the C-fragment of Oli or variantthereof or any of the other split intein C-fragments or variants thereofdiscussed below; the N-fragment of Sel(PC7942) or variant thereof canbind to the C-fragment of Sel(PC7942) or variant thereof or any of theother split intein C-fragments or variants thereof discussed below; theN-fragment of Ssp(PCC7002) or variant thereof can bind to the C-fragmentof Ssp(PCC7002) or variant thereof or any of the other split inteinC-fragments or variants thereof discussed below; the N-fragment of Telor variant thereof can bind to the C-fragment of Tel or variant thereofor any of the other split intein C-fragments or variants thereofdiscussed below; the N-fragment of Ter or variant thereof can bind tothe C-fragment of Ter or variant thereof or any of the other splitintein C-fragments or variants thereof discussed below; and theN-fragment of Tvu or variant thereof can bind to the C-fragment of Tvuor variant thereof or any of the other split intein C-fragments orvariants thereof discussed below.

The most widely used intein-based technology, expressed proteinligation, exploits cis-acting inteins to generate recombinant proteinα-thioester derivatives.(2) In principle, any split intein can beartificially fused and then utilized as a cis-splicing intein in thisapplication (1 in FIG. 4a ). Ultrafast split inteins are especiallyattractive in this regard due to their speed and efficiency. To testthis notion, artificially fused variants of Npu, Ava, and Mcht with anN-terminal ubiquitin domain were generated. To prevent splicing,premature C-terminal cleavage or undesired high levels of competinghydrolysis residues Asn137 and Cys+1 were mutated to Ala. Upon reactionwith the exogenous thiol sodium 2-mercaptoethanesulfonate (MESNa), thefused DnaE inteins were rapidly cleaved to generate the ubiquitinα-thioester, 4, in a few hours (FIGS. 4b ). By contrast, MESNa thiolysisof the commonly used MxeGyrA intein was not complete even after one dayunder identical conditions. The fused DnaE inteins were sufficientlyfast to allow for a one-pot thiolysis and native chemical ligationreaction with an N-terminal cysteine-containing fluorescent peptide, 5,to give semisynthetic protein 6 (FIG. 4c ). Furthermore, these inteinscould be used to efficiently generate α-thioesters of four otherstructurally unique proteins domains with different C-terminal aminoacid residues. These results demonstrate that fused versions of splitDnaE inteins will be of general utility for protein semisynthesis.

The rapid rate of thiolysis observed for the fused DnaE inteins hasmechanistic implications as well as practical ones. Without wishing tobe bound by theory, one possible explanation for their enhancedreactivity over the MxeGyrA intein is that these inteins drive theN-to-S acyl shift reaction more efficiently, generating a largerpopulation of the reactive linear thioester species 2 (FIG. 4a ). Thisthioester intermediate is generally thought to be transiently populatedin protein splicing, and to our knowledge, it has never been directlyobserved.(1) Surprisingly, when analyzing the ubiquitin-DnaE inteinfusions by reverse phase HPLC, two major peaks and a third minor peakwere often observed, all bearing the same mass. The relative abundanceof these species could be modulated by unfolding the proteins or bychanges in pH, and the two major species were almost equally populatedfrom pH 4-6 (FIG. 4d ). The major peaks most likely correspond to theprecursor amide, 1, and the linear thioester, 2, and the minor peak asthe tetrahedral oxythiazolidine intermediate. Importantly, only a singleHPLC peak was seen for the ubiquitin-MxeGyrA fusion under identicalconditions. These observations, along with the enhanced thiolysis rates,strongly support the notion that these DnaE inteins have ahyper-activated N-terminal splice junction.

Splicing activities in an entire family of split inteins has beencharacterized. Ultrafast protein trans-splicing may be the norm, ratherthan the exception, in this family. Furthermore, different split inteinshave varying degrees of tolerance for C-extein mutations, suggestingthat traceless protein splicing may be attainable by modestlyengineering any highly active intein. A thorough comparison of theactivities of a small family of homologous proteins can be used toidentify important non-catalytic positions that modulate activity.Finally, by artificially fusing split DnaE intein fragments, newconstructs have been provided for the efficient synthesis of proteinα-thioesters used in expressed protein ligation. These results willguide the development of improved protein chemistry technologies andshould lay the groundwork towards a more fundamental understanding ofefficient protein splicing.

Fusion Proteins of Split Intein N-Fragment

Disclosed herein are fusion proteins of a polypeptide and a split inteinN-fragment. As used herein, the term “polypeptide” refers to any aminoacid based polymer, interchangeable referred to as a “protein”throughout, and can include glycoproteins and lipoproteins. In somecases, the polypeptide is a polypeptide excreted from a cell (e.g., amammalian cell). In various cases, the polypeptide is an antibody or afragment thereof. The polypeptide can be any naturally occurring orsynthetic polypeptide of interest, including polypeptides having one ormore amino acid residues other than the 20 naturally occurring aminoacids.

In some cases, the polypeptide has a molecular weight of 45 kDa orgreater, 50 kDa or greater, 60 kDa or greater, 75 kDa or greater, 100kDa or greater, 120 kDa or greater, or 150 kDa or greater. Thepolypeptide can be, e.g., an antibody or a fragment thereof. In cases ofantibodies, the split intein N-fragment can be fused to one or both ofthe heavy chains, and/or to one or both of the light chains. In somecases, the polypeptide is a protein secreted from a cell, e.g., amammalian cell.

The split intein N-fragment comprises a sequence as shown in FIG. 5,e.g., Npu (SEQ ID NO: 1), Ssp (SEQ ID NO: 2), Aha (SEQ ID NO: 3), Aov(SEQ ID NO: 4), Asp (SEQ ID NO: 5), Ava (SEQ ID NO: 6), Cra(CS505) (SEQID NO: 7), Csp(CCY0110) (SEQ ID NO: 8), Csp(PCC8801) (SEQ ID NO: 9), Cwa(SEQ ID NO: 10), Maer(NIES843) (SEQ ID NO: 11), Mcht(PCC7420) (SEQ IDNO: 12), Oli (SEQ ID NO: 13), Sel(PC7942) (SEQ ID NO: 14), Ssp(PCC7002)(SEQ ID NO: 15), Tel (SEQ ID NO: 16), Ter (SEQ ID NO: 17), Tvu (SEQ IDNO: 18), or a variant thereof. In some cases, the spilt inteinN-fragment sequence comprises a sequence other than Npu (SEQ ID NO: 1)or Ssp (SEQ ID NO: 2), and in other cases, comprises a sequence otherthan Npu (SEQ ID NO: 1), Ssp (SEQ ID NO: 2), or Aha (SEQ ID NO: 3). Insome specific cases, the split intein N-fragment sequence comprises asequence of Ava (SEQ ID NO: 6), Cra (SEQ ID NO: 7), Csp(PCC8801) (SEQ IDNO: 9), Cwa (SEQ ID NO: 10) , Mcht(PCC7420) (SEQ ID NO: 12), Oli (SEQ IDNO: 13), Ter (SEQ ID NO: 17) and Tvu (SEQ ID NO: 18). In some cases, thesplit intein N-fragment has a sequence comprising a consensus sequenceof SEQ ID NO: 19:(CLSYDTElLTVEYGAVPIGKIVEENlECTVYSVDENGFVYTQPIAQWHDRGEQEVFEYCLEDGSTIRATKDHKFMTEDGEMLPIDEIFEQGLDLKQVKGLPD).

As used herein, a variant of a split intein N-fragment is a mutatedsplit intein N-fragment as disclosed herein that maintains the activityof the split intein N-fragment (e.g., its ability to bind to a splitintein C-fragment and/or catalyze nucleophilic attack of the polypeptidefused to it). Contemplated variants of a split intein N-fragmentsdisclosed herein include mutation of one or more C residues, except forCys1, to an aliphatic residue, such as an A, I, L, or F, or to a Sresidue. One such variant contemplated is a mutant Npu with Cys28 andCys59 mutated to Ser, SEQ ID NO: 20(CLSYETEILTVEYGLLPIGKIVEKRIESTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYSLEDGSURATKDHKFMTVDGQMLNDElFERELDLMRVDNLPN).

Mutated Split Intein C-Fragments and Binding to a Support

The split intein C-fragments disclosed herein are mutated from thenaturally occurring sequences to mutate the N137 and C+1 residues to aresidue other than Asn or Gln for N137 and a residue other than Cys forC+1 (SEQ ID NOs: 129-146). In some cases, the mutations at these twopositions are to a hydrophobic residue, e.g., not containing a free SHthiol (Cys), a carboxylic acid (Asp, Glu), or a base (Arg, His, Lys) orother undesired group (e.g., Asn, Gln) on the side chain. In variouscases, the two mutated aliphatic residues can be the same or differentand can be A, V, I, S, M, H, L, F, Y, G, or W or can be a unnatural(e.g., not encoded by genetic code) aliphatic amino acid residue such asnorleucine, 2-aminobutyric acid, nor-valine, 2-aminopentoic acid, or2-aminohexaanoic acid (SEQ ID NOs: 219-236). Specifically contemplatedare mutations where both residues are selected from A, I, V, L, Y, G andF (SEQ ID NOs: 309-326). In various cases, at least one of the twomutated residues is A.

Thus, provided herein is a mutated split intein C-fragment comprising amutation at N137 and Cys+1 of Npu (SEQ ID NOs: 129, 147, 165, 183, 201,219, 237, 255, 273, 291, 309, 327, 345, 363, 381 and 399), Ssp (SEQ IDNOs: 130, 148, 166, 184, 202, 220, 238, 256, 274, 292, 310, 328, 346,364, 382 and 400), Aha (SEQ ID NOs: 131, 149, 167, 185, 203, 221, 239,257, 275, 293, 311, 329, 347, 365, 383 and 401), Aov (SEQ ID NOs: 132,150, 168, 186, 204, 222, 240, 258, 276, 294, 312, 330, 348, 366, 384 and402), Asp (SEQ ID NOs: 133, 151, 169, 187, 205, 223, 241, 259, 277, 295,313, 331, 349, 367, 385 and 403), Ava (SEQ ID NOs: 134, 152, 170, 188,206, 224, 242, 260, 278, 296, 314, 332, 350, 368, 386 and 404),Cra(CS505) (SEQ lDNOs: 135, 153, 171, 189, 207, 225, 243, 261, 279, 297,315, 333, 351, 369, 387 and 405), Csp (CCY0110) (SEQ ID NOs: 136, 154,172, 190, 208, 226, 244, 262, 280, 298, 316, 334, 352, 370, 388 and406), Csp(PCC8801) (SEQ ID NOs: 137, 155, 173, 191, 209, 227, 245, 263,281, 299, 317, 335, 353, 371, 389 and 407), Cwa (SEQ ID NOs: 138, 156,174, 192, 210, 228, 246, 264, 282, 300, 318, 336, 354, 372, 390 and408), Maer(NIES843) (SEQ ID NOs: 139, 157, 175, 193, 211, 229, 247, 265,283, 301, 319, 337, 355, 373, 391 and 409), Mcht(PCC7420) (SEQ ID NOs:140, 158, 176, 194, 212, 230, 248, 266, 284, 302, 320, 338, 356, 374,392 and 410), Oli (SEQ ID NOs: 141, 159, 177, 195, 213, 231, 249, 267,285, 303, 321, 339, 357, 375, 393 and 411), Sel(PC7942) (SEQ ID NOs:142, 160, 178, 196, 214, 232, 250, 268, 286, 304, 322, 340, 358, 376,394 and 412), Ssp(PCC7002) (SEQ ID NOs: 143, 161, 179, 197, 215, 233,251, 269, 287, 305, 323, 341, 359, 377, 395 and 413), Tel (SEQ ID NOs:144, 162, 180, 198, 216, 234, 252, 270, 288, 306, 324, 342, 360, 378,396 and 414), Ter (SEQ ID NOs: 145, 163, 181, 199, 217, 235, 253, 271,289, 307, 325, 343, 361, 379, 397 and 415), or Tvu (SEQ ID NOs: 146,164, 182, 200, 218, 236, 254, 272, 290, 308, 326, 344, 362, 380, 398 and416), where the mutation at N137 and Cys+1 is a naturally occurring orunnatural hydrophobic residue. In some specific cases, at least one ofthe mutations is A (SEQ ID NOs: 183-200, 255-272, 327-344, and 345-416)and in more specific cases, both mutations are A (SEQ IS NOs: 399-416).

A variety of supports can be used. Generally, the solid support is apolymer or substance that allows for linkage of the split inteinC-fragment, optionally via a linker. The linker can be further aminoacid residues engineered to the C-terminus of the split inteinC-fragment or can be other known linkers for attachment of a peptide toa support. One contemplated linker is a small peptide −SGGC (SEQ ID NO:705), where the thiol of the C-terminal Cys can be used to attach thesplit intein C-fragment to the support. Thus, specifically contemplatedare mutated split intein C-fragments of the Npu, Ssp, etc. sequencesnoted above having a −SGGC peptide linker (SEQ ID NO: 705) (e.g.,specifying the residues starting at the N137 position: AAFN-SGGC) (SEQID NO:706). The length of a pepide linker can be modified to providevarying lengths and flexibility in any individual sitation (e.g., morethan 2 Gly residues). It will also be apparent that the C-terminusresidue of a peptide linker can be modified to introduce anappropriately reactive functional group to attach the split inteinC-fragment to a surface of choice (e.g., Lys to react via an amine, Cysto react via a thiol, or Asp or Glu to react via a carboxylic acid).Other, unnatural amino acid residues are also contemplated for use in apeptide linker to provide other functional group moieties to allow fordifferent attachment chemistry of the C-fragment to a support ofinterest (e.g., azide, alkynes, carbonyls, amino-oxy,cyano-benzothiazoles, tetrazoles, alkenes, alkyl-halides). The linkercan alternatively be a polymeric linker.

Based upon an analysis of the sequences of the highly active splitintein C-fragments investigated, a consensus sequence for the splitintein C-fragment is derived: SEQ ID NO: 707(VKIISRQSLGKQNVYDIGVEKDHNFLLANGLIASN), as well as a mutated versionwhere the N137 is mutated to other than Asn or Gln (SEQ ID NO:708), ormore specifically, N137 is mutant to a naturally occurring orunnaturally occurring hydrophobic residue, such as A, V, I, M, H, L, F,Y, G, S, H, or W or can be a unnatural (e.g., not encoded by geneticcode) aliphatic amino acid residue such as norleucine, 2-aminobutyricacid, nor-valine, 2-aminopentanoic acid, or 2-aminohexanoic acid (SEQ IDNO: 709). Specifically contemplated mutations at N137 for the consensussequences include A, I, V, L, Y, G, and F (SEQ ID NO: 710). Alsocontemplated is where N137 is mutated to A (SEQ ID NO: 711).

Further contemplated are variants of the consensus sequence having aresidue at the +1 position other than Cys (SEQ ID NOs: 712, 716, 720,and 724). More specifically the +1 position can be a naturally occurringor unnaturally occurring hydrophobic residue such as A, V, I, M, H, L,F, Y, G, S, H, or W or can be a unnatural (e.g., not encoded by geneticcode) aliphatic amino acid residue such as norleucine, 2-aminobutyricacid, nor-valine, 2-aminopentanoic acid, or 2-aminohexanoic acid (SEQ IDNOs: 713, 717, 721, and 725). Specifically contemplated are mutation at+1 position is selected from A, I, V, L, Y, G, and F (SEQ ID NOs: 714,718, 722, and 726). In various cases, at least one of the mutatedresidues of the consensus sequence is A (SEQ ID NOs: 715, 719, and 723).In some cases, the consensus C-fragment sequence has both mutations asAla (SEQ ID NO:727). Further contemplated is a consensus sequencecomprising FN at the +2 and +3 positions (SEQ ID NO:728-743. Alsocontemplated is a consensus sequence comprising a peptide linker forattachment to a solid support, and one embodiment is −SGGC at positions+4-+7 (SEQ ID NO: 744-759).

The split intein C-fragment or variant thereof as disclosed herein canbe attached to a solid support via a linker. In various cases, thelinker is a polymer, including but not limited to a water-solublepolymer, a nucleic acid, a polypeptide, an oligosaccharide, acarbohydrate, a lipid, or combinations thereof. It is not critical whatthe linker's chemical structure is, since it serves primarily as alinker. The linker should be chosen so as not to interfere with theactivity of the C-fragment. The linker can be made up of amino acidslinked together by peptide bonds. Thus, in some embodiments, the linkercomprises Y_(n), wherein Y is a naturally occurring amino acid or asteroisomer thereof and “n” is any one of 1 through 20. The linker istherefore can be made up of from 1 to 20 amino acids linked by peptidebonds, wherein the amino acids are selected from the 20naturally-occurring amino acids. In some cases, the 1 to 20 amino acidsare selected from Gly, Ala, Ser, Cys. In some cases, the linker is madeup of a majority of amino acids that are sterically un-hindered, such asGly.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —HN—(CH₂), —CO—, wherein s=2-20 can be used. These alkyl linkers mayfurther be substituted by any non-sterically hindering group such aslower alkyl (e.g., C₁ -C₆), halogen (e.g., Cl, Br), CN, NH₂, phenyl,etc.

Another type of non-peptide linker is a polyethylene glycol group, suchas: —HN—(CH₂)₂—(O—CH₂—CH₂)₂—O—CH₂—CO, wherein n is such that the overallmolecular weight of the linker ranges from approximately 101 to 5000,preferably 101 to 500.

In some cases, the linker has a length of about 0-14 sub-units (e.g.,amino acids).

In instances wherein the linker is a polynucleotide, the length of thelinker in various embodiments is at least about 10 nucleotides, 10-30nucleotides, or even greater than 30 nucleotides. In various aspects,the bases of the polynucleotide linker are all adenines, all thymines,all cytidines, all guanines, all uracils, or all some other modifiedbase.

In another embodiment, a non-nucleotide linker of the inventioncomprises a basic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds.Specific examples include those described by Seela and Kaiser, NucleicAcids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload andSchepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz,J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic AcidsRes. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991,10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al.,Biochemistry 1991, 30:9914; Arnold et al., International Publication No.WO 89/02439; Usman et al., International Publication No. WO 95/06731;Dudycz et al., International Publication No. WO 95/11910 and Ferentz andVerdine, J. Am. Chem. Soc. 1991, 113:4000, the disclosures of which areall incorporated by reference herein. A “non-nucleotide” further meansany group or compound that can be incorporated into a nucleic acid chainin the place of one or more nucleotide units, including either sugarand/or phosphate substitutions, and allows the remaining bases toexhibit their enzymatic activity. The group or compound can be abasic inthat it does not contain a commonly recognized nucleotide base, such asadenosine, guanine, cytosine, uracil or thymine, for example at the C1position of the sugar.

In various aspects, linkers contemplated include linear polymers (e.g.,polyethylene glycol, polylysine, dextran, etc.), branched chain polymers(see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al.,issued Sep. 15, 1981; 5,229,490 to Tam, issued Jul. 20, 1993; WO93/21259 by Frechet et al., published 28 Oct. 1993); lipids; cholesterolgroups (such as a steroid); or carbohydrates or oligosaccharides. Otherlinkers include one or more water soluble polymer attachments such aspolyoxyethylene glycol, or polypropylene glycol as described U.S. Pat.Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and4,179,337. Other useful polymers as linkers known in the art includemonomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol, as well as mixtures of these polymers.

In still other aspects, oligonucleotide such as poly-A or hydrophilic oramphiphilic polymers are contemplated as linkers, including, forexample, amphiphiles (including oligonucletoides).

Contemplated solid supports include resins, particles, and beads. Morespecific solid supports include polyhydroxy polymers, e.g. based onpolysaccharides, such as agarose, dextran, cellulose, starch, pullulan,or the like, and synthetic polymers, such as polyacrylic amide,polymethacrylic amide, poly(hydroxyalkylvinyl ethers),poly(hydroxyalkylacrylates) and polymethacrylates (e.g.polyglycidylmethacrylate), polyvinyl alcohols and polymers based onstyrenes and divinylbenzenes, and copolymers in which two or more of themonomers corresponding to the above-mentioned polymers are included.Specific solid supports contemplated include agarose, sepharose,cellulose, polystyrene, polyethylene glycol, derivatized agarose,acrylamide, sephadex, sepharose, polyethyleneglycol (PEG)-acrylamide,and polystyrene-PEG based supports. In some cases, the solid support canbe a resin such as p-methylbenzhydrylamine (pMBHA) resin (PeptidesInternational, Louisville, Ky.), polystyrenes (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), includingchloromethylpolystyrene, hydroxymethylpolystyrene andaminomethylpolystyrene, poly (dimethylacrylamide)-grafted styreneco-divinyl-benzene (e.g., POLYHIPE resin, obtained from Aminotech,Canada), polyamide resin (obtained from Peninsula Laboratories),polystyrene resin grafted with polyethylene glycol (e.g., TENTAGEL orARGOGEL, Bayer, Tubingen, Germany) polydimethylacrylamide resin(obtained from Milligen/Biosearch, California), or Sepharose (Pharmacia,Sweden). In various embodiments, the solid support can be a magneticbead, a glass slide, a glass bead, or a metal or inorganic particle(e.g., gold, silica, iron, or mixture thereof).

Methods of Purifying and Modifying Polypeptides

Site-specific modification of proteins is an invaluable tool to studythe molecular details of protein function (19). Moreover, its potentialfor the discovery and development of protein therapeutics has also beenrecently acknowledged (20). Several methods have been developed over theyears to generate site-specifically modified proteins; one of the mostwidely used is Expressed Protein Ligation (EPL), which has been appliedto many different proteins in a variety of studies to addressfundamental questions of protein function. EPL was first described in1998 (21), as an expansion to recombinant proteins of Native ChemicalLigation (NCL) (19,22), and it consists on the reaction between aC-terminal recombinant protein α-thioester with a synthetic peptidecontaining a Cys at its N-terminus through the formation of a new nativepeptide bond between the two fragments. The synthetic nature of the Cyscontaining peptide allows for the incorporation of almost any chemicalmodification into the protein of interest.

In order to apply EPL to any given protein the generation of proteinC-terminal thioesters in good yields and high purity is an absoluterequirement. A family of single turn-over enzymes, named inteins, hasbeen used since the dawn of EPL for the generation of such thioesters.Inteins are able to catalyze protein splicing, a naturally occurringpost-translational modification by which they excise themselves from thepolypeptide in which they are embedded, concomitantly forming a newpeptide bond between their flanking protein regions (23). Importantly,this reaction occurs via several protein α-thioesters, which can betrapped through a trans-thioesterification reaction with an exogenousthiol.

Inteins, such as GyrA or VMA, have been successfully harnessed toprepare a wide variety of protein thioesters. In order to isolate thedesired protein thioesters inteins are usually fused to affinity tagssuch as the chitin bidning domain or the hexa-His tag. However, despitethe notable success of this strategy, the reaction conditions requiredfor efficient thiolysis (reducing agents, large concentration of thiolsand long incubation times) affect the performance of such tags andsubsequent additional purification steps are often required to obtainthe desired pure product for ligation (24-26). Moreover, depending onthe identity of the C-terminal residue of the protein of interest,significant levels of in vivo premature cleavage can occur,significantly reducing the final product yield.

An ideal system should combine the thioester formation capabilities ofinteins with a built-in affinity purification strategy (fully compatiblewith the thiolysis reaction conditions) and reduced risk of prematurecleavage. Naturally split-inteins were investigated, which can perform areaction analogous to protein splicing but in which the intein itself issplit into two different polypeptides. Each of the two intein fragmentsare completely inactive by themselves but have a strong affinity foreach other and, upon binding, they adopt their splicing competent activeconformation and are able to carry out protein trans-splicing. Recently,an artificially split version of the DnaB intein has been reported forthe purification of unmodified proteins (27). Thus, a purificationstrategy is provided using naturally split-inteins instead and toharness them for the one pot purification and generation of recombinantprotein α-thioesters (FIG. 7) directly from cell lysates.

Due to the extremely fast reaction kinetics of naturally split inteins,several mutations were introduced to allow efficient thioester formationand minimize in vivo and in vitro undesired cleavage reactions.Specifically, both the C-intein C-terminal catalytic Asn137 and theCys+1 residues had to be mutated to Ala to prevent premature C- andN-terminal cleavage, respectively. Mutation to two sequential aliphaticresidues, natural or unnatural, is also expected to yield comparableresults as the AA mutation. Other mutated split intein C-fragments asdescribed above can be used in the described purification and /ormodification methods, and are specifically contemplated.

To develop a split-intein based purification and thioester formationstrategy the Npu split-intein was chosen, which is one of the fastestDnaE split-inteins previously known [10]. Initially the ability of splitNpu to generate protein thioesters in solution was tested by mixing themodel protein ubiquitin fused to NpuN with a mutant NpuC (Asn137 andCys+1 to Ala) in the presence (and absence) of the thiol MESNa. SDSPAGE,HPLC and MS analysis of the reactions showed the formation of thedesired ubiqutin C-terminal α-thioester in a few hours. Encouraged bythese results an affinity purification strategy was designed based onthe covalent immobilization of the NpuC intein mutant onto a solidsupport. The immobilized mutated NpuC could then be used to purify NpuNtagged proteins from complex mixtures and addition of an exogenous thiolwould cleave off the desired protein α-thioester, which would elute fromthe column in a highly purified form. Other split intein N-fragments asdescribed above can be used in the methods disclosed herein, and arespecifically contemplated.

An NpuC mutant (Asn¹³⁷ and Cys⁺¹ mutated to Ala, NpuC-AA (SEQ. ID NO:777) was prepared with a Cys residue at the C-terminus of its C-extein,which was used to immobilize the peptide onto a iodoacetyl resin. Withthe NpuC-AA affinity resin in hand, it was shown that several proteinC-terminal α-thioesters (Ubiquitin, MBP, PHPT1) could be easily producedand purified out of cell lysates (FIG. 8). HPLC and MS analysisconfirmed the formation of the desired protein thioesters with very lowlevels of undesired hydrolysis (FIG. 9). Recovery yields varied between75 and 95% and the NpuC-AA resin had a consistent loading capacity of3-6 mg of protein per mL. The utility of the α-thioester derivatives ofUb, MBP, and PHPT1 obtained from the column was demonstrated by ligatingeach of them to an N-terminal Cys-containing fluorescent peptide(CGK(Fl)) to give the corresponding semisynthetic products in excellentyield (FIG. 13). Importantly, one-pot thiolysis/ligation reactions canbe carried out, which provides a site-specifically modified proteindirectly from cell lysates without isolating the intermediate thioester(FIG. 14).

A concern when working with split-inteins (and also inteins) is theeffect of the flanking amino acid sequences on splicing and/or thiolysisactivity. Although the N-terminal junction is regarded as more toleranttowards deviations from the native N-extein residues it was important toevaluate the effect that the C-terminal amino acid of the protein ofinterest (−1 residue according to intein numbering conventions) wouldhave on the yields of thioester formation. A complete library ofUb-X-NpuN fusion proteins was constructed where the C-terminal Ubresidue (X) was varied from its native Gly to all other 19 proteinogenicamino acids. Proteins were expressed in E. coli and cell lysates,applied to the NpuC-AA affinity resin and purified. Protein yields wereestimated from the SDSPAGE analysis for each purification and hydrolysislevels from RP-HPLC and MS analysis of the elution fractions (FIG. 10).Results show similar trends to those known for non-split inteins, suchas GyrA (29), and most amino acids display high yields of cleavage afterovernight incubation with MESNa, the exceptions being Pro and Glu, forwhich recovery were 49 and 50%, respectively. As expected, the Asnα-thioester could not be isolated due to the well-known reaction of itsside-chain with the adjacent α-thioester to form a succinimide.

This purification strategy was very successful for the purification ofseveral soluble proteins under native conditions. However, proteinfragments required for EPL sometimes suffer from poor solubility andhigh toxicity and tend to accumulate in cellular inclusion bodies duringexpression. The Npu split-intein has been shown to retain a significantlevel of activity in the presence of denaturants (28), which suggestedthat this strategy would be compatible under such conditions. Using themodel Ub-NpuN protein fusion it was confirmed that both, binding to theNpuC-AA-resin and thioester formation, worked well in the presence of 2and 4 M urea. Similar levels of both, binding and thiolysis, wereobtained than in the absence of denaturant and same reaction conditions.

The system was next tested for the purification from inclusion bodies ofa fragment of the histone H2B. Preparation of site-specifically modifiedhistones using EPL is a topic of major interest due to their crucialrole in the understanding of epigenetic regulation. However, histonefragments are remarkably poorly behaved and the preparation of theirrecombinant C-terminal α-thioesters particularly challenging. AH2B(1-116) fragment fused to NpuN was expressed in E. coli and theinclusion bodies extracted with 6 M urea. The H2B-NpuN fusion wassubsequently diluted into a 3 M urea buffer and the correspondingC-terminal α-thioester generated concomitant with purification over theNpuC-AA affinity resin (FIG. 11). Due to its tendency of aggregation,very dilute protein solutions bound more efficiently to the resin, andalso longer reaction times were required for efficient thioestergeneration, obviously, these are parameters that would need to beoptimized on a protein to protein basis. Using these conditions,H2B(1-116) C-terminal thioester was obtained in excellent purity (>90%by RP-HPLC) and isolated yield (˜20 mg per L of culture). Thisrepresents a significant improvement over previous protocols whichafford less protein (4 mg per L of culture) and require the use ofmultiple chromatographic purification steps including RP-HPLC.Importantly, the H2B(1-116)-MES thioester obtained from the IntC-columncan be directly used in EPL reactions without further purification.Accordingly, the protein was successfully ligated to a syntheticH2B(117-125) peptide containing an acetylated Lys at position 120 toyield semi-synthetic H2B-K120Ac (FIG. 15).

The potential of this thioester formation purification strategy wasdemonstrated by applying it to the site-specific modification of amonoclonal antibody. Thus, specifically contemplated is a method ofpurifying an antibody by using a fusion protein of an antibody and asplit intein N-fragment as disclosed herein and a mutated split inteinC-fragment as disclosed herein.

The modification of antibodies is a field of intense research, speciallyfocused on the development of therapeutic antibody-drug-conjugates (30).The identity of the N-intein could have a significant effect on theexpression levels of its fusion to a given protein of interest. TheN-fragment of several of the fastest split DnaE inteins cross-reactedwith NpuC, allowing one to use any of them with the same NpuC basedaffinity column. Accordingly, the expression levels of a model antibody(αDEC, antibody against the DEC205 receptor) were tested and found thehighest levels of expression were obtained when αDEC was fused to theAvaN intein (FIG. 12A). αDEC-AvaN fusions were transfected into 293Tcells and after 4 days of culture the supernatants were collected andpurified over the NpuC-AA-column. The presence of a C-terminal thioesterin the purified αDEC was confirmed by reacting it with a shortfluorescent peptide with an N-terminal Cys residue (FIG. 12B and C). MSof the deglycosylated and reduced αDEC-fluorophore conjugate was used toconfirm its identity and SEC-MALS demonstrate the product wasmonodisperse and of the expected size for an IgG antibody.

Split-inteins can be engineered for the preparation of proteinα-thioesters and that the strong affinity between the two split-inteinfragments provides a powerful handle for their purification. Thegenerality of the approach is demonstrated by using it to generatehighly pure thioesters of both soluble (ubiqutin, MBP, PHPT) andinsoluble proteins (H2B fragment) as well as monoclonal antibodies(αDEC). Moreover, several N-inteins can be tested for optimal expressionlevels of the protein of interest and used with one single NpuC-column.

Thus, the split inteins disclosed herein can be used to purify andmodify a polypeptide of interest. A polypeptide of interest is providedin a fusion protein with a split intein N-fragment, e.g., via well-knownrecombinant protein methods. The fusion protein is then contacted with acorresponding split intein C-fragment under conditions that allowbinding of the N-fragment and C-fragment to form an intein intermediate.The split intein C-fragment can be bound to a support (e.g., a solidsupport such as a resin) or can subsequently (e.g., after binding to thesplit intein N-fragment to form the intein intermediate) be bound to asupport. This allows for the removal via washing of components that werein the mixture due to the recombinant protein synthesis, allowing thefusion protein to be isolated from the other components. Washes caninclude detergents, denaturing agents and salt solutions (e.g., NaCl).

Then, the intein intermediate can be reacted with a nucleophile torelease the polypeptide of interest from the bound N- and C-fragmentinteins wherein the C-terminus of the polypeptide is modified by thenucleophile added. The nucleophile can be a thiol to directed yield thepolypeptide as an α-thioester, which in turn can be further modified,e.g., with a different nucleophile (e.g., a drug, a polymer, anotherpolypeptide, a oligonucleotide), or any other moiety using thewell-known α-thioester chemistry for protein modification at theC-terminus. One advantage of this chemistry is that only the C-terminusis modified with a thioester for further modification, thus allowing forselective modification only at the C-terminus and not at any otheracidic residue in the polypeptide.

The nucleophile that is used in the methods disclosed herein either withthe intein intermediate or as a subsequent nucleophile reacting with,e.g., a α-thioester, can be any compound or material having a suitablenucleophilic moiety. For example, to form a α-thioester, a thiol moietyis contemplaed as the nucleophile. In some cases, the thiol is a1,2-aminothiol, or a 1,2-aminoselenol. An α-selenothioester can beformed by using a selenothiol (R-SeH). Alternative nucleophilescontemplated include amines (i.e. aminolysis to give amides directly),hydrazines (to give hydrazides), amino-oxy groups (to give hydroxamicacids). Additionally, the nucleophile can be a functional group within acompound of interest for conjugation to the polypeptide of interst(e.g., a drug to form a protein-drug conjugate) or could alternativelybear an additional functional group for subsequent known bioorthogonalreactions such as an azide or an alkyne (for a click chemistry reactionbetween the two function groups to form a triazole), a tetrazole, anα-ketoacid, an aldehyde or ketone, or a cyanobenzothiazole.

Additional aspects and details of the invention will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

EXAMPLES Materials

All buffering salts, isopropyl-P-D-thiogalactopyranoside (IPTG), andN,N-diisopropylethylamine (DIPEA) were purchased from Fisher Scientific(Pittsburgh, Pa.). Kanamycin sulfate (Kan), (β-Mercaptoethanol (BME),DL-dithiothreitol (DTT), sodium 2-mercaptoethanesulfonate (MESNa),ethanedithiol (EDT), Coomassie brilliant blue, N,N-dimethylformamide(DMF), Tetrakis(tiphenylphosphine)palladium(0) (Pd(PPh₃)₄),phenylsilane, triisopropylsilane (TIS), sodium diethyldithiocarbamatetrihydrate, and 5(6)-carboxyfluorescein were purchased fromSigma-Aldrich (St. Louis, Mo.). Tris(2-carboxyethyl)phosphinehydrochloride (TCEP) was purchased from Thermo Scientific (Rockford,Ill. Fmoc-Gly-OH, Fmoc-Lys(Alloc)-OH, and Boc-Cys(Trt)-OH were purchasdfrom Novabiochem (Laufelfingen, Switzerland). Piperidine was purchasedfrom Alfa Aesar (Ward Hill, Mass.). Dichloromethane (DCM) and rink amideresin were purchased from EMD Chemicals (Billerica, Mass.).1-Hydroxybenzotriazole hydrate (HOBt) was purchased from AnaSpec(Fremont, Calif.). Trifluoroacetic acid (TFA) was purchased fromHalocarbon (North Augusta, S.C.). Complete protease inhibitor tabletswere purchased from Roche Diagnostics (Mannheim, Germany).Nickel-nitrilotriacetic acid (Ni-NTA) resin was from Novagen (Gibbstown,N.J.). The QuikChange XL II site directed mutagenesis kit was fromAgilent (La Jolla, Calif.). DpnI and the Phusion High-Fidelity PCR kitwere from New England Biolabs (Ipswich, Mass.). DNA purification kits(QIAprep spin minikit, QIAquick gel extraction kit, QIAquick PCRpurification kit) were from Qiagen (Valencia, Calif.). Sub-cloningefficiency DH511 competent cells and One Shot BL21(DE3) chemicallycompetent E. coli were purchased from Invitrogen (Carlsbad, Calif.) andused to generate “in-house” high-competency cell lines. Oligonucleotideswere purchased from Integrated DNA Technologies (Coralville, Iowa). Thenew intein genes were generated synthetically and purchased from GENEWIZ(South Plainfield, N.J.). All plasmids used in this study were sequencedby GENEWIZ.

Criterion XT Bis-Tris gels (12%), Immun-blot PVDF membrane (0.2μm), andBradford reagent dye concentrate were purchased from Bio-Rad (Hercules,Calif.). 20× MES-SDS running buffer was purchased from BostonBioproducts (Ashland, Mass.). Mouse anti-myc monoclonal antibody (α-myc)was purchased from Invitrogen (Carlsbad, Calif.). Anti-His Tag, cloneHIS.H8 mouse monoclonal antibody (α-His6) was purchased from Millipore(Billerica, Mass.). Mouse HA.11 monoclonal antibody (α-HA) was purchasedfrom Covance (Princeton, N.J.). IRDye 800CW goat anti-Mouse IgGsecondary antibody (Licor mouse 800) and Licor Blocking Buffer werepurchased from LI-COR Biotechnology (Lincoln, Nebr.).

Equipment

Size-exclusion chromatography was carried out on an AKTA FPLC systemfrom GE Healthcare. Both preparative and analytical FPLC were carriedout on a Superdex 75 10/300 or S200 10/300 column. For all runs,proteins were eluted over 1.35 column volumes of buffer (flow rate: 0.5mL/min). Analytical RP-HPLC was performed on Hewlett-Packard 1100 and1200 series instruments equipped with a C18 Vydac column (5 μm, 4.6×150mm) at a flow rate of 1 mL/min. Preparative RP-HPLC was performed on aWaters prep LC system comprised of a Waters 2545 Binary Gradient Moduleand a Waters 2489 UV detector. Purifications were carried out on a C18Vydac 218TP1022 column (10 μM; 22×250 mm). All runs used 0.1% TFA(trifluoroacetic acid) in water (solvent A) and 90% acetonitrile inwater with 0.1% TFA (solvent B). For all runs, a two minute isocraticperiod in initial conditions was followed by a 30 minute linear gradientwith increasing buffer B concentration. Electrospray ionization massspectrometric analysis (ESI-MS) was performed on a Bruker DaltonicsMicrOTOF-Q II mass spectrometer. In vivo intein activity assays werecarried out on a VersaMax tunable microplate reader from MolecularDevices. Cells were lysed using an S-450D Branson Digital Sonifier.Western blots and coomassie-stained in vitro splicing assay gels wereimaged on a LI-COR Odyssey Infrared Imager. Fluorescentfluorescein-containing gels were imaged using the GE ImageQuant LAS 4000imager.

Compilation of the DnaE Sequence Library and Sequence Aanalysis

Protein sequences of the split DnaE inteins were obtained from the NEBInBase1. This list consisted of 23 entries as of May 2011. Of theseentries, two were discarded from the study as they did not have aC-intein sequence: Csp(PCC7822) and Nosp(CCY9414). Two pairs of inteinshad identical sequences: Nsp(PCC7120) with Asp (these are most likelythe same organism with two different names) and Sel(PCC6301) withSel(PC7942). Thus, Nsp(PCC7120) and Sel(PCC6301) were removed from thelibrary. The Mcht(PCC7420) and Oli C-intein sequences were identical,but both inteins were kept in the library as their N-intein sequenceswere different. In the InBase, the Aov intein had an “X” at position 87in place of an absolutely conserved isoleucine (I), so 187 was utilizedat this position. The plasmid for the kanamycin resistance assaysbearing the Csp(PCC7424) intein proved to be unstable and yielded highlyvariable results; thus this intein was excluded from the analyses. Thefinal library contained 18 inteins, Table 1.

TABLE 1 DnaE Intein Name Genus Species Strain Npu Nostoc punctiformePCC73102 Ssp Synechocystis species PCC6803 Aha Aphanothece halophyticaAov Aphanizomenon ovalisporum Asp Anabaena species PCC7120 Ava Anabaenavariabilis ATCC29413 Cra(CS505) Cylindrospermopsis raciborskii CS-505Csp(CCY0110) Cyanothece species CCY0110 Csp(PCC8801) Cyanothece speciesPCC8801 Cwa Crocosphaera watsonii WH 8501 Maer(NIES843) Microcystisaeruginosa NIES-843 Mcht(PCC7420)- Microcoleus chthonoplastes PCC7420 2Oli Oscillatoria limnetica Solar Lake Sel(PC7942) Synechococcuselongatus PC7942 Ssp(PCC7002) Synechococcus species PCC7002 TelThermosynechococcus elongatus BP-1 Ter-3 Trichodesmium erythraeum IMS101Tvu Thermosynechococcus vulcanus

Given the high homology of DnaE intein sequences, the N- and C-inteinswere manually aligned using the multiple alignment software Jalview2.All N-intein sequences were “left-justified” to align the first cysteineresidue, and the variable N-intein tail region was not aligned. AllC-intein sequences were “right-justified” to align the C-terminalasparagine. The residue numbering used in this study is based on thenumbering for the NMR structure of a fused Npu intein (PDB code 2KEQ).Thus, the variable N-intein tail region after residue 102 (the lastresidue of NpuN) is excluded from the numbering, as is the N-terminalmethionine of the C-intein. The C-intein numbering starts at 103, exceptfor the Tel and Tvu inteins, which have a gap at this position and startat 104. For the sequence logos (FIG. 6), the N- and C-intein alignmentswere each separated into two alignments based on high and low activity.The high activity sequence logos were comprised of Cwa, Cra(CS505),Csp(PCC8801), Ava, Npu, Csp(CCY0110), Mcht(PCC7420), Maer(N1ES843), Asp,Oli, and Aha (which was included based on the high activity of the C120Gmutant). The low activity sequence logos were comprised of Aov, Ter,Ssp(PCC7002), Tvu, Tel, Ssp, and Sel(PC7942). The sequence logos weregenerated using WebLogo. (4) Heat maps were generated using thestatistical computing and graphics program “R”.

Cloning of Plasmids for In Vivo Screening

The aminoglycoside phosphotransferase (KanR) and Npu gene fragments werecloned into a pBluescript KS (+) vector between KpnI and SacIrestriction sites as previously described (36,37). This constructcontained the following architecture:

[KanR promoter]-[RBS]-[myc-KanRN]-[IntN]-[iRBS]-[IntC]-[CFN-KanRC] wherethe KanR promoter is the constitutive promoter found in mostkanamycin-resistant plasmids, RBS is a common E. coli ribosomal bindingsite, iRBS is an intervening ribosomal binding site preceded by alinker, myc encodes for a c-myc epitope tag (EQKLISEEDL) (SEQ ID NO:760), KanRN and KanRC are fragments of the KanR protein, and IntN andIntC are split intein fragments. An analogous Ssp plasmid was alsoconstructed as previously described (36,37). These plasmids are referredto as myc-KanR-NpuDnaE-Split and myc-KanR-SspDnaE-Split. To generate thescreening vectors for the remaining split inteins, synthetic genes weredesigned and purchased from GENEWIZ containing the followingarchitecture:

[5′ overhang]-[IntN]-[iRBS]-[IntC]-[3′ overhang] where the 5′ and 3′overhangs were the exact 39 bp found upstream of NpuN and 25 bp founddownstream of NpuC, respectively, in the myc-KanR-NpuDnaE-Split plasmid.For all inteins, the purchased gene sequences were codon-optimized withthe default E. coli codon usage table generated based on all E. colicoding sequences in GenBank8. The synthetic genes were received in pUC57vectors.

To clone the screening plasmids, the entire synthetic gene was amplifiedwith Phusion High-Fidelity Polymerase using primers annealing to the 5′and 3′ overhangs. The resulting megaprimer was inserted into themyc-KanR plasmid in place of Npu by overlap-extension PCR with Phusionpolymerase (39). This resulted in 18 homologous plasmids containingidentical backbones, promotors, and KanR genes, but with differentcodon-optimized intein genes. The plasmids are named as:myc-KanR-XyzDnaE-Split (where Xyz indicates the intein name as given inTable 1). Specific point mutations were made to various inteins using aQuikChange Site-Directed Mutagenesis kit with the standard recommendedprotocol.

In Vivo Screening of Relative Intein Aactivities

96-well plate assay: Intein activity-coupled kanamycin resistance (KanR)assays were conducted in 96-well plate format as previously described(36,37). Typically, plasmids were transformed into 15 μL of sub-cloningefficiency DH5α cells by heat shock, and the transformed cells weregrown for 18 hours at 37° C. in 3 mL of Luria-Bertani (LB) media with100 μg/mL of ampicillin (LB/amp). The over-night cultures were diluted250-fold into LB/amp solutions containing 8 different kanamycinconcentrations (150 μL per culture). The cells were grown at 30° C. on a96-well plate, monitoring optical density (OD) at 650 nm every 5 minutesfor 24 hours while shaking for one minute preceding each measurement.The endpoint of this growth curve (typically in the stationary phase)was plotted as a function of kanamycin concentration to visualize thedose-response relationship and fitted to a variable-slope dose-responseequation to determine IC₅₀ values.

${OD}_{Obs} = {{OD}_{Min} + \frac{\left( {{OD}_{Max} - {OD}_{Min}} \right)}{1 + 10^{\lbrack{{({{\log \; {IC}_{50}} - {\log {\lbrack{Kan}\rbrack}}})} \cdot {HillSlope}}\rbrack}}}$

In each regression analysis, typically three or four independent doseresponse curves were collectively fit to the equation above using theGraphPad Prism software. In each fit, 0D_(Min) was fixed to thebackground absorbance at 650 nm, and all other parameters were allowedto vary. The reported error bars for the IC₅₀ bar graphs (FIG. 1b )represent the standard error in the best-fit IC₅₀ value from three orfour collectively fit dose-response curves.

Western blot analysis of in vivo splicing: For the western blotanalyses, DH5α cells were transformed with the assay plasmidsidentically as for the 96-well plate setup and grown for 18 hours at 37°C. while shaking. The overnight cultures were used to inoculate 3 mL offresh LB/amp at a 1:300 dilution, and the cells were incubated at 30° C.for 24 hours. The ODs of the 30° C. cultures were measured at 650nm toassess relative bacterial levels, then 150 μL of each culture wastransferred to an Eppendorf tube and centrifuged at 17,000 rcf for 2minutes. The supernatant was aspirated off, and the cell pellets wereresuspended/lysed in ˜200 μL of 2× SDS gel loading dye containing 4% BME(the resuspension volumes were varied slightly to normalize fordifferences in OD). The samples were boiled for 10 minutes, thencentrifuged at 17,000 rcf for 1 minute. Each sample (5 μL) was loadedonto a 12% Bis-Tris gel and run in MES-SDS running buffer. The proteinswere transferred to PVDF membrane in Towbin transfer buffer (25 mM Tris,192 mM glycine, 15% methanol) at 100V for 90 minutes. Membranes wereblocked with 4% milk in TBST, then the primary antibody (α-myc, 1:5000)and secondary antibody (Licor mouse 800, 1:15,000) were sequentiallyapplied in 4% milk in TBST. The blots were imaged using the LicorOdyssey scanner.

Cloning of Plasmids for In Vitro Splicing Assays

Ub-IntN plasmids: The N-intein expression plasmids were derived from apreviously described NpuN plasmid, pMR-Ub-NpuN(WT) (36,37). This plasmidencoded for the following protein sequence:

(SEQ ID NO: 761) MHHHHHHGGMQIFVKTLIGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQELIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGGGGGKFAEY CLSYETEILTVEYGLLPIGKIVEICRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGELIRATKDHICFMTVDGQMLPIDEIFERELDLMRVDNLPN where the NpuN sequence is given in bold, the immediate native localextein residues are underlined, and these residues are preceded byHis₆-Ub with a Gly₄ linker. Significant in vivo proteolysis waspreviously observed during expression of this construct, so this plasmidwas modified using QuikChange to remove the Gly₄ sequence. The resultingplasmid, pMR-Ub-NpuN-ΔGly₄ was used as the template for all otherUb-IntN plasmids and encoded for the following protein sequence:

(SEQ ID NO: 762) MHHHHHHGGMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQELIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGKFAEY CLSYETEILTVEYGLLPIGKIVEICRIECTVYSVDNNGNIYTQPVAQVIHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

All other IntN plasmids were cloned using overlap-extension PCR togenerate Ub-IntN fusion genes in homologous plasmids in a tracelessmanner (39). Specifically, N-intein genes were amplified by Phusionpolymerase from the synthetic gene plasmids using primers with overhangsthat anneal to the plasmid sequences surrounding NpuN inpMR-Ub-NpuN-ΔGly₄. The resulting megaprimer was then used to insert thenew N-intein gene in place of NpuN to generate a new plasmid calledpMR-Ub-IntN that was identical to the NpuN plasmid except for theN-intein gene.

IntC-SUMO plasmids: The C-intein plasmids were all derived from apreviously described NpuC plasmid, pET-NpuC(WT)-SUMO (37). This plasmidencoded for the following protein sequence:

(SEQ ID NO: 763) MGSSHHHHHHGENLYFQ|GIKIATRKYLGKQNVYDIGVERDHNFALKNGF IASNCFNSGLVPRGSASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGYPYDVPDYAwhere the NpuC sequence is given in bold, and the immediate native localextein residues are underlined, followed by a linker sequence andSUMO-HA. This construct is preceded by a His₆-tag and a tobacco etchvirus (TEV) protease recognition sequence. The TEV protease cleavagesite is indicated by “|” and leaves behind a glycine residue in place ofan N-terminal IntC methionine.

All other IntC plasmids were cloned using overlap-extension PCR togenerate IntC-SUMO fusion genes in homologous plasmids in a tracelessmanner (39). Specifically, C-intein genes were amplified by Phusionpolymerase from the synthetic gene plasmids using primers with overhangsthat anneal to the plasmid sequences surrounding NpuC inpET-NpuC(WT)-SUMO. The resulting megaprimer was then used to insert thenew C-intein gene in place of NpuC to generate a new plasmid calledpET-IntC-SUMO that was identical to the NpuC plasmid except for theC-intein gene.

Purification of Proteins for In Vitro Splicing Assays

Over-expression and purification of Ub-IntN constructs (except Ub-CwaN):E. coli BL21(DE3) cells transformed with each N-intein plasmid weregrown in 1 L of LB containing 100 μg/mL of ampicillin at 37° C. untilOD₆₀₀=0.6. The cells were then cooled down to 18° C., and expression wasinduced by addition of 0.5 mM IPTG for 16 hours at 18° C. Afterharvesting the cells by centrifugation (10,500 rcf, 30 min), the cellpellets were transferred to 50 mL conical tubes with 5 mL of lysisbuffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, 2 mM BME, pH 8.0)and stored at −80° C. The cell pellets were resuspended by adding anadditional 15 mL of lysis buffer supplemented with Complete proteaseinhibitor cocktail. Cells were lysed by sonication (35% amplitude, 8×20second pulses separated by 30 seconds on ice). The soluble fraction wasrecovered by centrifugation (35,000 rcf, 30 min). The soluble fractionwas mixed with 2 mL of Ni-NTA resin and incubated at 4° C. for 30minutes. After incubation, the slurry was loaded onto a fitted column.After discarding the flow-through, the column was washed with 5 columnvolumes (CV) of lysis buffer, 5 CV of wash buffer 1 (lysis buffer with20 mM imidazole), and 3 CV of wash buffer 2 (lysis buffer with 50 mMimidazole). The protein was eluted with elution buffer (lysis bufferwith 250 mM imidazole) in four 1.5 CV elution fractions. The wash andelution fractions were analyzed by SDS-PAGE.

After enrichment over the Ni-NTA column, the proteins were purified bygel filtration. The wash and elution fractions were all treated with 50mM DTT for 30 minutes on ice. For well-expressing proteins, the firstelution fraction was then directly injected on an S75 10/300 gelfiltration column (3× 1 mL injections) and eluted over 1.35 CV infreshly prepared, degassed splicing buffer (100 mM phosphates, 150 mMNaCl, 1 mM DTT, 1 mM EDTA, pH 7.2). For the more dilute, low-yieldingproteins, typically the 50 mM imidazole wash fraction and the first twoelution fractions were pooled and concentrated four-fold to 3 mL. Then,the concentrated protein was purified by gel filtration identically tothe high-yielding constructs. FPLC fractions were analyzed by SDS-PAGE,and the purest fractions were pooled and analyzed by analytical gelfiltration, analytical RP-HPLC, and mass spectrometry. The concentrationof pure proteins were determined by UV A280 nm and by the Bradfordassay.

Over-expression and purification of Ub-CwaN: The Ub-CwaN protein did notexpress well in the soluble fraction, and all of the enriched proteinwas aggregated, as observed by gel filtration analysis. Thus, afterexpression, cell lysis, and fractionation, as described above, theprotein was extracted from the insoluble fraction of the lysate asfollows. First, the lysate pellet was resuspended in 20 mL of Tritonwash buffer (lysis buffer with 0.1% Triton X-100) and incubated at roomtemperature for 30 minutes. The Triton wash was centrifuged at 35,000rcf for 30 minutes, and the supernatant was discarded. Next, the pelletwas resuspended in 20 mL of lysis buffer containing 6 M urea, and themixture was incubated overnight at 4° C. The mixture was centrifuged at35,000 rcf for 30 minutes, and then the supernatant was mixed with 2 mLof NiNTA resin. The Ni column was run identically as for the nativepurifications described above, except that every buffer had a backgroundof 6 M urea. Following enrichment over a Ni-NTA column, the 50 mMimidazole wash and the first two elution fractions were pooled anddiluted to 0.2 mg/mL. The diluted protein was refolded into lysis buffer(without urea) by step-wise dialysis removal of the urea at 4° C. Theprotein was concentrated four-fold to 3 mL and immediately purified bygel filtration as indicated for the native purifications above. The pureprotein was analyzed by analytical gel filtration, analytical RP-HPLC,and mass spectrometry. Note that this construct was highly susceptibleto aggregation. When re-folded at 2 mg/mL rather than 0.2 mg/mL, lessthan 10% of the obtained protein was monomeric, whereas more diluterefolding yielded roughly 50% monomeric protein. The obtained proteinwas 80% monomeric, and the monomer to aggregate ratio did not changeafter 24 hours of storage at 4° C. The concentration of pure protein wasdetermined by the Bradford assay.

Over-expression and purification of IntC-SUMO constructs: E. coliBL21(DE3) cells transformed with each C-intein plasmid were grown in 1 Lof LB medium containing kanamycin (50 μg/mL) at 37° C. until OD₆₀₀=0.6.Then, expression was induced by addition of 0.5 mM IPTG for 3 hours at37° C. The cells were lysed, and the desired protein was enriched overNi-NTA resin identically as for the natively purified Ub-IntN proteins.The AvaC-SUMO and Csp(PCC8801)C-SUMO proteins did not express well at37° C., so the proteins were re-expressed by induction at 18° C. for 16hours. For each protein, the 50 mM imidazole wash and the first twoelution fractions were pooled and dialyzed into TEV cleavage buffer (50mM phosphate, 300 mM NaCl, 5 mM imidazole, 0.5 mM EDTA, 0.5 mM DTT, pH8.0) then treated with 40 μg of His-tagged TEV protease overnight atroom temperature. The cleavage was confirmed by RP-HPLC/MS, after whichthe reaction solution was incubated with Ni-NTA resin at roomtemperature for 30 min. The flow-through and two 1.5 CV washes with washbuffer 1 were collected and pooled. The protein was then concentrated to3-4 mL, injected onto the S75 10/300 gel filtration column (3× 1 mLinjections), and eluted over 1.35 CV in freshly prepared, degassedsplicing buffer (100 mM phosphates, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH7.2). FPLC fractions were analyzed by SDS-PAGE, and the purest fractionswere pooled and analyzed by analytical gel filtration, analyticalRP-HPLC, and mass spectrometry. The concentration of pure protein wasdetermined by UV _(280 nm) and by the Bradford assay.

Usage and storage of the Ub-IntN and IntC-SUMO constructs: All of thepurified proteins were stored at 4° C. and used within two days forsplicing assays with their cognate IntC-SUMO. The remaining protein (2vol. eq.) was mixed with splicing buffer containing 60% glycerol (1 vol.eq.) to yield a 20% glycerol stock that was aliquoted and flash frozenin liquid N₂. The protein aliquots were stored at -80° C. The proteinswere fully functional after thawing on ice and could be flash-frozen andre-thawed at least once without detectable loss of function.

In Vitro Splicing Assays

Kinetic assay procedure: For a typical assay, individual protein stocksolutions of Ub-IntN and IntC-SUMO constructs were prepared in filteredsplicing buffer (100 mM phosphate, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH7.2) at 2× the final concentration (e.g. 2.0 μM stock solution for a 1.0μM reaction). 1 mM TCEP was added (from a pH-neutralized 100 mM stocksolution) to each protein solution, and the proteins were incubated at30° C. or 37° C. for 5 min depending on the reaction temperature. Toinitiate a reaction, the N- and C-intein were mixed at equal volumes(i.e. equimolar ratios). A typical reaction volume was 300 μL and wascarried out in an Eppendorf tube on a heat block. During the reaction,20 μL aliquots of the reaction solution were removed at the desired timepoints and quenched in 20 μL of 2× concentrated SDS gel loading dye onice to afford a final quenched solution with 40 mM Tris (˜pH 7.0), 10%(v/v) glycerol, 1% (w/v) SDS, 0.02% (w/v) bromophenol blue, and 2% (v/v)BME. For each reaction, an artificial zero time point was taken bymixing equivalent amounts of starting materials directly into thequencher solution. Samples were boiled for 10 minutes then centrifugedat 17,000 rcf for 1 minute. Aliquots of starting materials and timepoints (15 μL) were loaded onto Bis-Tris gels and run in MES-SDS runningbuffer. The gels were Coomassie-stained then imaged using the LicorOdyssey scanner.

Note that for the reactions with a CGN C-extein sequence, no BME wasused in the quencher solution. Furthermore, before boiling the samples,each sample was treated with 1 μL of 2 N HC1. After boiling and coolingthe samples, they were treated with 1μL of 2 N NaOH. This procedureprevented undesired hydrolysis or thiolysis of the branchedintermediate.

Determination of kinetic parameters: To determine reaction rates, eachlane of a gel was analyzed using the Licor Odyssey quantificationfunction or ImageJ. Given the close proximity of the starting materialbands, these bands were typically integrated together. To normalize forloading error, the integrated intensity of each band in a lane wasexpressed as a fraction intensity of the total band intensity in thatlane (which remained relatively constant between lanes). Thesenormalized intensities were plotted as a function of time, and data fromthree independent reactions were collectively fit to first-order rateequations using the GraphPad Prism software:

For reactant depletion: Y=S·(e ^(−k) ^(obs) ^(·))+Z

For product formation: Y=Y _(max)·(1−e^(−k) obs ^(·t))

Y is the fractional intensity of a species, t is time in minutes, S is ascaling factor for reactant depletion (allowed to vary), Z indicates thefraction of reactant remaining at the reaction endpoint (allowed tovary), Y_(max) is a scaling factor for product formation, and k_(obs) isthe observed first-order rate constant for the splicing reaction(allowed to vary). Half-lives were calculated from the best-fit valuefor the first-order rate constant:

$t_{1/2} = \frac{\ln \; 2}{k_{obs}}$

For reactions with no detectable side product formation, the rate ofproduct (Ub-SUMO) and IntN formation were consistent with the rate ofstarting material depletion.

Western blot analysis of reactions: Western blots of the zero time pointand reaction endpoint were carried out to confirm the identities of theobserved bands. The quenched time points from the reactions describedabove were loaded onto 12% Bis-Tris gels (5 μL per sample, two identicalgels) and run in MES-SDS running buffer. The resolved proteins weretransferred from the gel onto PVDF membrane in CAPS transfer buffer (10mM N-cyclohexyl-3-aminopropanesulfonic acid, 10% (v/v) methanol, pH10.5) at 100 V for 60 minutes. Membranes were blocked with LicorBlocking Buffer, then the primary antibody (α-His₆, 1:3000, or α-HA,1:25,000) was applied in Licor Blocking Buffer. The secondary antibody(Licor mouse 800, 1:15,000) was applied in 4% milk in TBST. The blotswere imaged using the Licor Odyssey scanner. Blots from the 30° C. and37° C. reactions were virtually identical.

HPLC/MS analysis of Npu-CGN and Cra(CS505)-CGN reactions

For the HPLC/MS analysis of Npu-CGN and Cra(CS505)-CGN, individualprotein stock solutions of Ub-IntN and IntC-CGN-SUMO were prepared infiltered splicing buffer (100 mM phosphate, 150 mM NaCl, 1 mM DTT, 1 mMEDTA, pH 7.2) at 8.0 μM. 1 mM TCEP was added (from a pH-neutralized 100mM stock solution) to each protein solution, and the proteins wereincubated at 30° C. for 5 min. To initiate a reaction, the N- andC-intein were mixed at equal volumes (i.e. equimolar ratios) andincubated at 30° C. During the reaction, 90 μL aliquots of the reactionsolution were removed at the desired time points and quenched in 30 μI.,of a quenching solution (6 M guanidine hydrochloride with 4%trifluoroacetic acid). 100 μI., of each quenched time point wereinjected onto an analytical C18 RP-HPLC column and eluted over a 25-73%buffer B gradient in 30 minutes, preceded by a two minute isocraticphase in 25% buffer B (see Equipment section for column and runningbuffer specifications). At different time points, various HPLC peakswere collected and their identities were confirmed by mass spectrometry.The IntC-(Ub)SUMO species were identified by MS, verifying branchedintermediate formation and depletion.

Kinetic Modeling

When comparing the Npu, Cra(CS505), and Cwa reactions in the presence ofCGN, higher amounts of cleaved ubiquitin (i.e. N-extein cleavage) wereobtained than splice product, despite the fact that the rate of theformer was slower than the latter. This observation is inconsistent withN-extein cleavage and splice product formation only occurring from thebranched intermediate, since in this scenario splicing and cleavagewould be competing first-order reactions occurring from the samereactant (the branched intermediate), leading to more splice productthan cleavage (the opposite of that observed). In an attempt toreconcile these observations, a series of kinetic modeling simulationswere carried out. All modeling was carried out using the kineticmodeling applet from BPReid (40). Themodels have three basic assumptionsabout the splicing pathway:

-   1. The forward and reverse reactions in the first equilibrium are    fast. In addition, the position of this equilibrium lies slightly    towards the amide.-   2. The second equilibrium is also fast and should have K_(eq) close    to 1, since both intermediates are cysteinyl thioesters.-   3. For fast inteins, the rate of branched intermediate resolution    (k₅) is on the same order    , of magnitude as the rates of the first two reversible steps,    whereas the cleavage rates from L (k₆) and B (k₇) are relatively    slow. For slow inteins, branched intermediate resolution (k₅) is    also slow, on the same order of magnitude as the cleavage.

With these assumptions, six scenarios were devised that assess how therelative rates of cleavage and branched intermediate resolution and theequilibrium between the linear and branched intermediates could affectthe rates and extents of formation of the cleavage and spliced products.For slow inteins, such as those bearing exogenous C-extein residues, therate of branched intermediate resolution is similar to the rate ofN-extein cleavage. Under these circumstances, three factors areimportant:

-   1. The relative rates of cleavage from L versus B (k₆ vs. k₇).-   2. The relative rates of branched intermediate resolution versus    cleavage (k₅ vs. k₆+k₇).-   3. Most importantly, the rates of exchange between the linear and    branched intermediates (k₃/k₄).    These analyses suggest not only that cleavage should be occurring    both from the linear and branched intermediate, but also that    cleavage at the linear intermediate may be favored.    Protein Thiolysis and Ligation from Fused DnaE Inteins and MxeGyrA

Solid-phase synthesis and purification of H-Cys-Gly-Lys(Fluorescein)-NH₂(CGK-Fluorescein): Fmoc-based solid phase peptide synthesis (SPPS) wasused to produce a peptide with the sequenceH-Cys-Gly-Lys(Fluorescein)-NH₂. The peptide was synthesized on Rinkamide resin at a 0.2 mmol scale as follows: 20% piperidine in DMF wasused for Fmoc deprotection using a one minute equilibration of the resinfollowed by a 20 minute incubation. After Fmoc deprotection, amino acidswere coupled using DIC/HOBt as activating agents. First, the amino acid(1.1 mmol) was dissolved in 50:50 DCM:DMF (2 mL) and was activated withDIC (1.0 mmol) and HOBt (1.2 mmol) at 0° C. for 15 minutes. The mixturewas added to the N-terminally deprotected resin and coupled for 10minutes at room temperature.

After the cysteine was coupled, the lysine side chain was deprotected bytreatment with Pd(Ph₃)₄ (0.1 eq.) and phenylsilane (25 eq.) in dry DCMfor 30 minutes. The peptidyl resin was washed with DCM (2×5 mL) and DMF(2×5 mL) followed by two washes with 0.5% DIPEA in DMF (v/v) and twowashes with 0.5% sodium diethyldithiocarbamate trihydrate in DMF (w/v)to remove any remaining traces of the Pd catalyst.5(6)-Carboxyfluorescein was then coupled to the lysine side chain usingthe DIC/HOBt activation method overnight at room temperature. Finally,the peptide was cleaved off the resin using 94% TFA, 1% TIS, 2.5% EDT,and 2.5% H₂O (6.5 mL) for one hour. After cleavage, roughly half of theTFA was evaporated under a stream of nitrogen. The crude peptide wasprecipitated with cold ether and washed with cold ether twice. Finally,the peptide was purified by RP-HPLC on C18 prep column over a 15-80%buffer B gradient in 40 minutes. The purified peptide was analyzed byanalytical RP-HPLC and ESI-MS to confirm its identity. Note that noattempt was made to separately isolate the 5-carboxyfluorescein and6-carboxyfluorescein conjugates, thus the peptide is a mixture of thesetwo isomers.

Cloning of Ub-Intein fusions: All Ub-Intein fusions were cloned into amodified pDCB1 vector from NEB containing ubiquitin in which a His₆-tagand stop codon were inserted between the MxeGyrA intein and the chitinbinding domain. This resulted in a plasmid, pTXB1-Ub-MxeGyrA-ATEA-H₆that encodes for the following protein, called calledUb-MxeGyrA-ATEA-H₆:

(SEQ ID NO: 764) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGCITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATEAHHHHHHin which the intein sequence for MxeGyrA (N198A) is shown in bold,preceded by ubiquitin and followed by the endogenous local C-exteinsequence (underlined) and a His₆-tag.

This plasmid was modified to replace the MxeGyrA intein with a fused Npuintein. First, the myc-KanR-NpuDnaE-Split plasmid was modified byQuikChange to remove the iRBS sequence separating the NpuN and NpuCgenes. The resulting plasmid, myc-KanR-NpuDnaE-Fused, was then used as atemplate to amplify megaprimers bearing the fused Npu intein withoverhangs homologous to the sequences surrounding MxeGyrA in themodified pDCB1 vector. The Npu gene with the N137A mutation was insertedin place of MxeGyrA using overlap-extension PCR with the Phusionpolymerase. (39) Importantly, this construct was modified to include thenative C-extein residues of Npu (CFN) instead of those for MxeGyrA(TEA). The resulting plasmid, pTXB1-Ub-NpuDnaE-ACFN-H₆ encoded for thefollowing protein:

(SEQ ID NO: 765) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASACFNHHHHHHThis fusion showed substantial in vivo hydrolysis of ubiquitin whenexpressed in E. coli. Thus, it was further modified using QuikChangemutagenesis by mutating the +1 cysteine to alanine, generating theplasmid pTXB1-Ub-NpuDnaE-AAFN-H₆. This plasmid encoded for the followingprotein (Ub-NpuDnaE-AAFN-H₆) that was used for in vitro thiolysisexperiments:

(SEQ ID NO: 766) MQIFVKILIGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRILSDYNIQKESTLHLVLRLRGGCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNIKIATRKYLGKQNVYDIGVERDHNFA LKNGFIASA AFNHHHHHHThe pTXB1-Ub-AvaDnaE-AAFN-H₆ and pTXB1-Ub-MchtDnaE-AAFN-H₆ plasmids,encoding for the following protein sequences (Ub-AvaDnaE-AAFN-H₆ andUb-MchtDnaE-AAFN-H₆, respectively), were cloned analogously by modifyingthe pTXB1 -Ub-NpuDnaE-AAFN-H₆ plasmid.

Ub-AvaDnnE-AAFN-H6 (SEQ ID NO: 767)MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGCLSYDTEVLTVEYGEVPIGEIVDKGIECSVESIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPEIKIASRKFLGVENVYDIGVGRDHNFF VKNGLIASA AFNHHHHHHUb-MchtDnaE-AAFN-H6 (SEQ ID NO: 768)MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGCLSYDTQILTVEYGAVAIGEIVEKQIECTVYSVDENGYVYTQPIAQWHNRGEQEVFEYLLEDGATIRATKDHKFMTDEDQMLPIDQIFEQGLELKQVEVLQPVFVKIVRRQSLGVQNVYDIGVEKDHN FCLASGEIASAAFNHHHHHHAs a control for the removal of the +1 Cys residue in the DnaE inteinconstructs, the +1 Thr residue was mutated from thepTXB1-Ub-MxeGyrA-ATEA-H₆plasmid by QuikChange mutagenesis to yield theplasmid pTXB1-Ub-MxeGyrA-AAEA-H₆, encoding for the proteinUb-MxeGyrA-AAEA-H₆.

(SEQ ID NO: 769) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGCITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQATADELTDGRFYYAKVASVTDAGVQPVYS LRVDTADHAFITNGFVSHAAEAHHHHHH

Cloning of additional fusions to fused DnaE inteins: Several otherproteins were fused to AvaDnaE or MchtDnaE to test the sequencedependence on thiolysis from these inteins. The proteins utilized werethe N-terminal SH3 domain of human Grb2 (AAs 1-55+/−an exogenousC-terminal Gly), the SH2 domain of human Abl kinase (AAs 122-217), eGFP,and the catalytic domain of human PARP1 (AAs 657-1015). All plasmidswere cloned using the aforementioned methods to yield plasmids encodingthe following proteins:

SH3-AvaDnaE-AAFN-H₆ (SEQ ID NO: 770)MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMCLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPEIKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASAAFNHHHHHH SH3-GLy-AvaDnaE-AAFN-H₆(SEQ ID NO: 771) MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMGCLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPEIKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASAAFNHHHHHH SH3-MchtDnaE-AAFN-H₆(SEQ ID NO: 772) MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMCLSYDTQILTVEYGAVAIGEIVEKQIECTVYSVDENGYVYTQPIAQWHNRGEQEVFEYLLEDGATIRATKDHKFMTDEDQMLPIDQIFEQGLELKQVEVLQPVFVKIVRRQSLGVQNVYDIGVEKDHNFCLASGEIASAAFNHHHHHH SH2-AvaDnaE-AAFN-H₆(SEQ ID NO: 773) MLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPACLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPEIKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASAAFNHHHHHH eGFP-AvaDnaE-AAFN-H₆(SEQ ID NO: 774) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKCLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPEIKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASAAFNHHHHHH  PARPc-AvaDnaE-AAFN-H6 (SEQ ID NO: 775)MVNPGTKSKLPKPVQDLIKMIFDVESMKKAMVEYEIDLQKMPLGKLSKRQIQAAYSILSEVQQAVSQGSSDSQILDLSNRFYTLIPHDFGMKKPPLLNNADSVQAKAEMLDNLLDIEVAYSLLRGGSDDSSKDPIDVNYEKLKTDIKVVDRDSEEAEIIRKYVKNTHATTHNAYDLEVIDIFKIEREGECQRYKPFKQLHNRRLLWHGSRTTNFAGILSQGLRIAPPEAPVTGYMFGKGIYFADMVSKSANYCHTSQGDPIGLILLGEVALGNMYELKHASHISKLPKGKHSVKGLGKTTPDPSANISLDGVDVPLGTGISSGVNDTSLLYNEYIVYDIAQVNLKYLLKLKFNFKTSLWCLSYDTEVLTVEYGFVPIGEIVDKGIECSVFSIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKATKDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPEIKIASRKFLGVENVYDIGVGRDHNFFVKNGLIASAAFNHHHHHH 

Purification of various Protein-Intein fusions: E. coli BL21(DE3) cellstransformed with each Protein-Intein fusion plasmid were grown in 1 L ofLB medium containing ampicillin (100 μg/mL) at 37° C. until OD₆₀₀=0.6.Then, expression was induced by addition of 0.5 mM IPTG and incubationfor 3 hours at 37° C. or incubation for 16 hours at 18° C. All Ubfusions were expressed at 37° C., the eGFP fusion was expressed at 18°C., and the SH3, SH2, and PARI³ _(C) fusions were expressed at bothtemperatures. After harvesting the cells by centrifugation (10,500 rcf,30 min), the cell pellets were transferred to 50 mL conical tubes with 5mL of lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, NoBME, pH 8.0) and stored at −80° C. The cell pellets were resuspended byadding an additional 15 mL of lysis buffer supplemented with Completeprotein inhibitor cocktail. Cells were lysed by sonication (35%amplitude, 8×20 second pulses separated by 30 seconds on ice). Thesoluble fraction was recovered by centrifugation (35,000 rcf, 30 min).The soluble fraction was mixed with 2 mL of Ni-NTA resin and incubatedat 4° C. for 30 minutes. After incubation, the slurry was loaded onto afitted column. After discarding the flow-through, the column was washedwith 5 column volumes (CV) of lysis buffer, 5 CV of wash buffer 1 (lysisbuffer with 20 mM imidazole), and 3 CV of wash buffer 2 (lysis bufferwith 50 mM imidazole). The protein was eluted with elution buffer (lysisbuffer with 250 mM imidazole) in four 1.5 CV elution fractions. The washand elution fractions were analyzed by SDS-PAGE with loading dyecontaining no thiols. The cleanest fractions were pooled and treatedwith 10 mM TCEP for 20 minutes on ice. Then, the solution was injectedon an S75 or S200 10/300 gel filtration column (2×1 mL injections), andeluted over 1.35 CV in thiolysis buffer (100 mM phosphates, 150 mM NaCl,1 mM EDTA, 1 mM TCEP, pH 7.2). The FPLC fractions were analyzed bySDS-PAGE with loading dye containing no thiols, and the purest fractionswere pooled and analyzed by analytical RP-HPLC and mass spectrometry.The concentration of pure protein was determined by UV A_(280 nm).

Thiolysis of Ub-Intein fusions and ligation of ubiquitin to a smallfluorescent peptide: For each Ub-Intein fusion protein, four reactionswere carried on a 100 μL scale at 30° C. In the first reaction tomonitor background hydrolysis, the fusion protein (50 μM) was incubatedin thiolysis buffer (100 mM phosphate, 150 mM NaCl, 1 mM EDTA, 1mM TCEP,pH 7.2) supplemented with freshly added TCEP (an additional 5 mM). Inthe second and third reactions, the protein was incubated identically asfor the first reaction, except that each reaction had either 100 mMMESNa or 1 mM CGK-Fluorescein. In the fourth reaction, both MESNa andthe peptide were added. At various time points, 5μL of reaction solutionwere removed and quenched in 30 μL 2× SDS loading dye containing nothiols. As time points were collected, they were stored at −20° C. untilthe end of the reaction. After the reaction, the 35 μL quenched timepoints were thawed, treated with 1μL of a 1 M TCEP stock solution,boiled for 10 minutes, and centrifuged at 17,000 rcf for 1 minute. Timepoints (5 μL) were loaded onto 12% Bis-Tris gels and run in MES-SDSrunning buffer. The gels were first imaged on a fluorescence imager tovisualize the Ub-CGK-Fluorescein ligation product. Then the gels werecoomassie-stained and imaged using the Licor Odyssey scanner. Inaddition, the reaction endpoints were quenched by 20-fold dilution inH₂O with 0.1% TFA and injected on an analytical C18 RP-HPLC column. Themixture was separated over a 2 minute isocratic phase in 0% B followedby a 0-73% B linear gradient in 30 minutes. The major peaks werecollected and analyzed by MS.

Thiolysis of SH3-, SH2-, eGFP-, and PARP_(c)-Intein fusions: Thiolysisreactions with several other proteins fused to the AvaDnaE and MchtDnaEfused inteins were carried out analogously to the ubiquitin reactionsdescribed above. In a typical reaction, carried out on a 300 μL scale at30° C., 10 μM fusion protein was treated with 5 mM TCEP in thiolysisbuffer then incubated in the presence or absence of MESNa (either 100 mMor 200 mM) added from a pH-adjusted 1 M stock solution. At various timepoints, aliquots (15 μL) of the reaction solution were quenched in 30μLof 2× SDS gel loading dye containing no thiols and stored at −20° C.until the end of the reaction. After the reaction, the 45 μL quenchedtime points were thawed, treated with 1μL of a 1 M TCEP stock solution,boiled for 10 minutes, and centrifuged at 17,000 rcf for 1 minute. Timepoints (15 μL) were loaded onto 12% Bis-Tris gels and run in MES-SDSrunning buffer. Then the gels were coomassie-stained and imaged usingthe Licor Odyssey scanner. In addition, the reaction endpoints werequenched by 4-fold dilution in H₂O with 0.1% TFA and injected on ananalytical C18 RP-HPLC column. The mixture was separated over a 2 minuteisocratic phase in 0% B followed by a 0-73% B linear gradient in 30minutes. The product peaks were collected and analyzed by MS.

Observation of the Linear Thioester Intermediate in Fused DnaE Inteins

For Npu, Ava, and Mcht fusions to ubiquitin, three peaks were visiblefor the purified protein when directly injected onto a C18 RP-HPLCcolumn from a neutral buffer. These peaks all had the same mass of thedesired protein. When diluted 20-fold in H₂O containing 0.1% TFA (pH 2)and incubated for at least two hours at room temperature, the first twopeaks merged into the third peak (FIG. 4d ). The same observation couldnot be made for MxeGyrA under identical conditions. To further confirmthat an equilibrium between the precursor amide and linear thioester wasoccuring, the Ub-NpuDnaE-AAFN-H₆ protein was diluted 20-fold inthiolysis buffer containing 1% SDS. Before boiling, the two major peakswere visible. After boiling for 10 minutes, when the protein wasunfolded, the first major peak partially converged into the second majorpeak, suggesting that the latter was the amide, which should be morestable in the unfolded intein. Additional evidence that the three peakswere in equilibrium came from pH titrations. The protein was diluted20-fold into citric acid/phosphate buffers ranging from pH 2 to pH 8,incubated at room temperature for 3-4 hours, then analyzed by HPLC overa 30-73% B gradient in 30 minutes (FIG. 4d ). The relative abundance ofthe three species was modulated and showed a bell-shaped pH dependence,similar to the activities of enzymes containing multiple ionizablefunctional groups in their active sites.

In addition to observing the desired protein mass from all threeobserved HPLC peaks, the presence of a −18 Da species was observed inthe first two peaks. This mass change is characteristic of a dehydrationreaction, and such a reaction has been previously reported by Mootz et.al. for a mutant form of the SspDnaB intein that cannot efficientlycatalyze the initial N-to-S acyl shift. (41) Specifically, thetetrahedral intermediate of the forward and reverse acylation reactionscan undergo acid-catalyzed dehydration to yield a thiazoline sideproduct. For Mootz and co-workers, this species was an irreversibleside-product for their mutant intein under normal reaction conditions,and it lead to low yields. In the systems herein, where the DnaE inteinscan react to completion, this species is either an artifact ofacidification during RP-HPLC or it is fully reversible under normalreaction conditions. It is noteworthy that the observation of thethiazoline by MS further validates the presence of detectable levels ofthe tetrahedral intermediate in the present reaction mixtures.

For the DnaE intein fusions to proteins other than ubiquitin, similarHPLC profiles were observed with multiple peaks at neutral pH, howeverthe ratios of the three peaks varied depending on the sequence.Additionally, for sequences more similar to the endogenous “A-E-Y” DnaEN-extein (such as the SH3 fusions with an “I-E-M” sequence), substantialaccumulation of a dehydrated product (as much as 50% by HPLC/MS) wasseen, similar to that observed by Mootz et. al. for the split SspDnaBintein. (41) For these constructs, this species appears to accumulateduring protein expression resulting in a mixture of “trapped”(dehydrated) and “free” (native, hydrated) fusion protein. Upon additionof MESNa at neutral pH, the “free” protein rapidly undergoes thiolysisto yield the desired product, and the “trapped” protein slowlyrehydrates and is also thiolyzed to yield the same desired product.Thus, in the reaction progress curves, a “burst” phase was observedfollowed by a slower phase. Importantly, the accumulation of dehydratedfusion protein could be reduced by expression at lower temperatures (18°C. instead of 37° C.), and these reactions could be driven faster andcloser to completion by increasing the MESNa concentration from 100 mMto 200 mM MESNa. In addition, it is noteworthy that for the SH3thiolysis reaction, the MchtDnaE intein was substantially more efficientthat the AvaDnaE intein, suggesting that different fused DnaE inteinsmay be preferable depending on the protein of interest.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

REFERENCES

-   (1) Mills, K. V.; Perler, F. B. Protein Pept. Lett. 2005, 12, 751-5.-   (2) Vila-Perelló, M.; Muir, T. W. Cell 2010, 143, 191-200.-   (3) Southworth, M.; Amaya, K.; Evans, T.; Xu, M.; Perler, F.    Biotechniques 1999, 27, 110-120.-   (4) Amitai, G.; Callahan, B. P.; Stanger, M. J.; Belfort, G.;    Belfort, M. Proc. Natl. Acad. Sci. USA 2009, 106, 11005-10.-   (5) Zettler, J.; Schütz, V.; Mootz, H. D. FEBS Lett. 2009, 583,    909-14.-   (6) Iwai, H.; Züger, S.; Jin, J.; Tam, P.-H. FEBS Lett. 2006, 580,    1853-8.-   (7) Perler, F. B. Nucleic Acids Res. 2002, 30, 383-4.-   (8) Caspi, J.; Amitai, G.; Belenkiy, O.; Pietrokovski, S. Mol.    Microbiol. 2003, 50, 1569-77.-   (9) Dassa, B.; Amitai, G.; Caspi, J.; Schueler-Furman, O.;    Pietrokovski, S. Biochemistry 2007, 46, 322-330.-   (10) Chen, L.; Zhang, Y.; Li, G.; Huang, H.; Zhou, N. Anal. Biochem.    2010, 407, 180-7.-   (11) Martin, D. D.; Xu, M. Q.; Evans, T. C. Biochemistry 2001, 40,    1393-402.-   (12) Lockless, S. W.; Muir, T. W. Proc. Natl. Acad. Sci. USA 2009,    106, 10999-1004.-   (13) Shah, N. H.; Vila-Perelló, M.; Muir, T. W. Angew. Chem. Int.    Ed. Engl. 2011, 50, 6511-5.-   (14) Oeemig, J. S.; Aranko, A. S.; Djupsjobacka, J.; Heinamaki, K.;    Iwai, H. FEBS Lett. 2009, 583, 1451-1456.-   (15) Du, Z.; Liu, Y.; Ban, D.; Lopez, M. M.; Belfort, M.; Wang,    C. J. Mol. Biol. 2010, 400, 755-67.-   (16) Appleby-Tagoe, J. H.; Thiel, I. V.; Wang, Y.; Wang, Y.;    Mootz, H. D.; Liu, X.-Q. J. Biol. Chem. 2011, 286, 34440-7.-   (17) Busche, A. E. L.; Aranko, A. S.; Talebzadeh-Farooji, M.;    Bernhard, F.; Dötsch, V.; Iwai, H. Angew. Chem. Int. Ed. Engl. 2009,    48, 6128-31.-   (18) Dhar, T.; Mootz, H. D. Chem. Commun. 2011, 47, 3063-5.-   (19) Pellois J-P, Muir T W: Current Opinion in Chemical Biology    2006, 10:487-491.-   (20) Cheriyan M, Perler F B: Adv Drug Deliv Rev 2009.-   (21) Muir T W, Sondhi D, Cole P A: Proc. Natl. Acad. Sci. U.S.A    1998, 95:6705-6710.-   (22) Dawson P E, Muir T W, Clark-Lewis I, Kent S B: Science 1994,    266:776-779.-   (23) Evans T C J R T C, Xu M-Q: Chem Rev 2002, 102:4869-4884.-   (24) Wu Y-W, Oesterlin LK, Tan K-T, Waldmann H, Alexandrov K, Goody    R S: Nat Chem Biol 2010, 6:534-540.-   (25) Frutos S, Goger M, Giovani B, Cowburn D, Muir T W: Nature    chemical biology 2010, 6:527-533.-   (26) lsen S K, Capili A D, Lu X, Tan D S, Lima C D:Nature 2010,    463:906-912.-   (27) Lu W, Sun Z, Tang Y, Chen J, Tang F, Zhang J, Liu J-N: Journal    of Chromatography A 2011, 1218:2553-2560.-   (28) Zettler J, Schatz V, Mootz H D: FEBS Letters 2009, 583:909-914.-   (29) Southworth M W, Amaya K, Evans T C, Xu M Q, Perler F B:    BioTechniques 1999, 27:110-114, 116, 118-120.-   (30) Carter P J: Exp Cell Res 2011, 317:1261-1269.-   (31) Perler, F. B. Nucleic Acids Res. 2002, 30, 383-4.-   (32) Waterhouse, A. M.; Procter, J. B.; Martin, D. M. A.; Clamp, M.;    Barton, G. J. Bioinformatics 2009, 25, 1189-91.-   (33) Oeemig, J. S.; Aranko, A. S.; Djupsjöbacka, J.; Heinämäki, K.;    Iwai, H. FEBS Lett. 2009, 583, 1451-1456.-   (34) Crooks, G. E.; Hon, G.; Chandonia, J.-M.; Brenner, S. E. Genome    Res. 2004, 14, 1188-90.-   (35) R Development Core Team; R Foundation for Statistical    Computing: Vienna, Austria, 2011.-   (36) Lockless, S. W.; Muir, T. W. Proc. Natl. Acad. Sci. USA 2009,    106, 10999-1004.-   (37) Shah, N. H.; Vila-Perelló, M.; Muir, T. W. Angew. Chem. Int.    Ed. 2011, 50, 6511-5.-   (38) Stothard, P. BioTechniques 2000, 28, 1102, 1104.-   (39) Bryksin, A. V.; Matsumura, I. BioTechniques 2010, 48, 463-5.-   (40) Reid, B. P.; B P Reid: Hanover, N.H., 2009.-   (41) Schwarzer, D.; Ludwig, C.; Thiel, I. V.; Mootz, H. D.    Biochemistry 2012, 51, 233-42.-   (42) Sun, P.; Ye, S.; Ferrandon, S.; Evans, T. C.; Xu, M.-Q.;    Rao, Z. J. Mol. Biol. 2005, 353, 1093-105

1.-34. (canceled)
 35. A method comprising (a) contacting (1) a fusionprotein comprising a polypeptide and a DnaE split intein N-fragment or avariant thereof, and (2) a DnaE split intein C-fragment where Asn 137 ismutated to a residue other than Asn or Glu, or a variant thereof,wherein contacting is performed under conditions that permit binding ofthe split intein N-fragment to the split intein C-fragment to form anintein intermediate; and (b) contacting the intein intermediate with anucleophile to form a conjugate of the protein and the nucleophile. 36.The method of claim 35, wherein the DnaE split intein N-fragmentcomprises a sequence selected from the group consisting of SEQ ID NO:1-19 or a variant thereof.
 37. The method of claim 35, wherein the DnaEsplit intein N-fragment comprises a sequence selected from the groupconsisting of SEQ ID NO: 20-38 or a variant thereof.
 38. The method ofclaim 35, wherein the DnaE split intein C-fragment comprises a sequenceselected from the group consisting of SEQ ID NO: 57-74 or a variantthereof.
 39. The method of claim 35, wherein the DnaE split inteinN-fragment comprises a sequence selected from the group consisting ofSEQ ID NO: 1-19 and the DnaE split intein C-fragment comprises asequence selected from the group consisting of SEQ ID NO: 57-74.
 40. Themethod of claim 35, wherein the split intein C-fragment is bound to asupport.
 41. The method of claim 40, wherein the support comprises abead, a resin, a particle or a slide.
 42. The method of claim 35,wherein the fusion protein is in a whole cell lysate.
 43. The method ofclaim 42, further comprising washing the intein intermediate to separatethe intein intermediate from components of the whole cell lysate. 44.The method of claim 35, wherein the fusion protein is from a cellsupernatant.
 45. The method of claim 44, further comprising washing theintein intermediate to separate the intein intermediate from componentsof the cell supernatant.
 46. The method of claim 35, wherein thepolypeptide has a molecular weight of 40 kDa or greater.
 47. The methodof claim 46, wherein the polypeptide is an antibody, or fragmentthereof.
 48. The method of claim 35, wherein the polypeptide is secretedfrom a cell.
 49. The method of claim 35, further comprising isolatingthe conjugate.
 50. The method of claim 35, wherein the nucleophilecomprises a second polypeptide, an oligonucleotide, a nanoparticle, adrug, or a polymer.
 51. The method of claim 35, wherein the nucleophilecomprises a thiol and the conjugate comprises a thioester.
 52. Themethod of claim 51, wherein the thiol is 2-mercaptoethansulfonate, analkyl thiol, or an aryl thiol.
 53. The method of claim 51, furthercomprising reacting the thioester with a second nucleophile to form asecond conjugate.
 54. The method of claim 53, wherein the secondnucleophile comprises an amine, a hydrazine, or an amino-oxy moiety.